Operator Manual - VWR

SmartCycler II

Operator Manual

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ii SmartCycler Life Sciences Operator ManualD1819 Rev D

Copyright 1999-2005 by Cepheid All rights reserved. Printed in USAThis manual contains information protected by copyright. No part of this manual may be photo-copied or reproduced in any form without prior written consent from Cepheid.

Trademark InformationSmartCycler® and I-CORE® are registered trademarks of Cepheid. SmartCycler® and I-CORE® are not to be used in any type of promotion or advertising without permission from Cepheid.

All other trademarks are the property of their respective owners.

Cepheid DisclaimerCepheid makes no warranties, express or implied, including without limitation the implied warran-ties of merchantability and fitness for a particular purpose, regarding the SmartCycler® System. Cepheid does not warrant, guarantee or make any representations regarding the use or the results of the use of SmartCycler® System in terms of its correctness, accuracy, reliability, currentness or otherwise, and the entire risk as to the results and the performance of SmartCycler® System is assumed by you. The exclusion of implied warranties is not permitted in some states, therefore the above exclusion may not apply to you.

In no event will Cepheid, its directors, officers, employees or agents be liable to you for any con-sequential, incidental or indirect damages (including damages for loss of business profits, busi-ness interruption, loss of business information, and the like) arising out of the use or inability to use SmartCycler® System, even if Cepheid has been advised of the possibility of such damages. Because some states do not allow the exclusion or limitation of liability for consequential or inci-dental damages, the above may not apply to you. Cepheid's liability to you for actual damages from any cause whatsoever, and regardless of the form of the action (whether in contract, tort [including negligence], product liability or otherwise), will be limited to $50.

Notice to PurchaserThis real-time thermal cycler is licensed for use in research, diagnostics (except in diagnosis and monitoring of HIV and HCV infections) and all other applied fields under Applera's European Patent No. EP 0 872 562, Japanese Patent No. JP 3136129 and patents pending. No rights are con-veyed expressly, by implication or estoppel to any patents on real-time methods, including but not limited to 5' nuclease assays, or to any patent claiming a reagent or kit. Applied Biosystems does not guarantee the performance of this instrument or endorse its use in any application.

Authorized Thermal CyclerThis instrument, Serial No. __________, is an Authorized Thermal Cycler. Its purchase price includes the up-front fee component of a license under the non-U.S. counterparts of United States Patents Nos. 4,683,195, 4,683,202 and 4,965,188 owned by F. Hoffmann-La Roche Ltd, cov-ering the Polymerase Chain Reaction (“PCR”) process, to practice the PCR process for internal research and development using this instrument. The running royalty component of that license may be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents. This instrument is also an Authorized Thermal Cycler for use with applications licenses available from

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Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Purchase of this product does not itself convey to the purchaser a complete license or right to perform the PCR process. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

No rights are conveyed expressly, by implication or estoppel to any patents on real-time methods, including but not limited to 5’ nuclease assays, or to any patent claiming a reagent or kit.

Applied Biosystems does not guarantee the performance of this instrument.

Cepheid SmartCycler® System Software License AgreementLicense Grant: Subject to the terms and conditions herein, Cepheid grants Customer a non-exclusive, non-transferable license (the “License”) to use one (1) copy of the Cepheid Smart-Cycler System Software program (the “Software”) on the computer provided with the Smart-Cycler System and connected to the SmartCycler processing block(s) and (1) copy on a second computer that is identical to the computer provided with the SmartCycler System, to be used only for SmartCycler data analysis and to make one (1) copy for back-up purposes. Other than expressly provided herein, Customer shall not copy, modify, duplicate, translate, disassemble, or decompile the Software without Cepheid's prior written consent.

License Type: Single-user Product. Customer shall use the Software only on the computer provided with the SmartCycler System or a second computer identical to the computer pro-vided with the SmartCycler System. If the computer is not identical to the one provided with the SmartCycler System, Cepheid will not guarantee performance and cannot provide tech-nical support for problems arising from the use of a different computer. Customer may not use this single-user product on a network.

Copyright: The ideas and expressions thereof contained in the Software are confidential and proprietary information and trade secrets of Cepheid that are disclosed to Customer in confi-dence. Customer shall not cause or permit decompilation, disassembly, or reverse engineer-ing of the Software or disclosure, copying, display, loan, publication, transfer of possession (whether by sales, exchange, gift, operation of law or otherwise) or other dissemination of the Software and related documentation, in whole or part, to any third party without the prior written consent of Cepheid. The Software and its related documentation are owned by Cepheid and protected by United States copyright laws and international treaty provisions. Customer shall not alter or remove any copyright, trade secret, patent, trademark, propri-etary and/or other legal notices contained on or in the Software and related documentation.

Ownership: Cepheid is the exclusive owner of the Software, related documentation and physical media, and of all copyright, trade secret, patent, trademark and other intellectual or industrial property rights therein. Physical media and copies of the Software, whether in dis-kette, tape, paper or other forms provided by Cepheid, shall remain the property of Cepheid, and such copies are deemed to be on loan to Customer during the term of the License granted hereby. Customer expressly acknowledges that no title to or ownership of the Soft-ware, or any copy or portion thereof, is transferred to Customer hereby.

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Support: This License entitles Customer to the use of toll free telephone support as provided by Cepheid.

Termination: This License is effective until terminated. Cepheid may terminate this License if Customer fails to comply with any of the terms or conditions of this License or of the Ceph-eid System Agreement. If this License is terminated, Customer must destroy all copies of the Software and its related documentation.

Warranty Information: CEPHEID SPECIFICALLY DISCLAIMS ALL OTHER WARRANTIES INCLUD-ING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND OF FIT-NESS FOR A PARTICULAR PURPOSE. Cepheid shall not be liable for incidental or consequential damages resulting from use of the Software. In particular, Cepheid does not warrant that the Software will meet Customer's requirements or that the operation of the Software will be uninterrupted or error free. In the event that the Software media is defec-tive, Cepheid will replace the defective media at no cost to Customer.

Government Customers: For Government customer, the Software is commercial computer software subject to restricted rights under FAR 52.227-19 (C) (1, 2).

Limitation of Liability: The Software is licensed on an “as is” basis. This means the entire risk as to the quality and performance of the Software is on Customer. IN NO EVENT WILL CEPH-EID BE LIABLE FOR ANY LOSS, DAMAGE, OR LIABILITY, INCLUDING, WITHOUT LIMITATION, ANY DIRECT, INDIRECT, SPECIAL, INCIDENTAL OR CONSEQUENTIAL DAMAGES INCURRED BY CUSTOMER OR ANY OTHER PERSON AS A RESULT OF, OR RELATED TO, THE DELIVERY, USE, OR PERFORMANCE OF THE CEPHEID SmartCycler SOFTWARE OR ANY BREACH OF THE WAR-RANTIES CONTAINED HEREIN, WHETHER DUE TO THE NEGLIGENCE OF CEPHEID OR OTHER-WISE. Some states do not allow the exclusion or limitation of incidental or consequential damages, in which case the above limitation or exclusion may not apply to Customer.

SmartCycler Processing Block Limited WarrantyCepheid warrants that the SmartCycler Processing Block (the Instrument) (i) is free from defects in material and workmanship and (ii) conforms to Cepheid's published specifications for the Instrument. This Warranty is for a period of 12 months from the date of shipment to the Purchaser (the Warranty Period). During the Warranty Period, if the Instrument's hard-ware is found to be defective or if the Instrument fails to meet specifications, Cepheid will repair or replace the Instrument so that it meets specifications, at a site determined by Cep-heid at Cepheid's expense. This Warranty is limited and applies only to new products manu-factured by Cepheid.

Cepheid does not warrant any defects in the Instrument caused by (i) improper installation, removal or testing, (ii) Purchaser’s failure to provide a suitable operating environment for the Instrument, (iii) use of the Instrument for purposes other than that for which they were designed, (iv) unauthorized attachments, (v) unusual physical or electrical stress, (vi) modifi-cations or repairs done by other than Cepheid or a Cepheid authorized service provider, or (vii) any other abuse, misuse, or neglect of the Instrument.

Purchaser's exclusive remedy for any defective Instrument is limited to the repair or replace-ment of any defective Instrument. If Cepheid cannot or does not repair or replace a defective

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Instrument, Cepheid will remove the Instrument and return the purchase price for the defec-tive Instrument. This warranty extends to Purchaser only, and not to Purchaser's customers or other third parties, except as agreed to in writing by Cepheid.

Even if Cepheid cannot or does not repair or replace any defective Instrument and Pur-chaser's exclusive remedy fails of its essential purpose, Cepheid's entire liability shall in no event exceed the purchase price for any defective Instrument, and Cepheid shall have no lia-bility for general, consequential, incidental or special damages arising from a defect in the Instrument.

THERE ARE NO WARRANTIES WHICH EXTEND BEYOND THE FACE HEREOF. CEPHEID DIS-CLAIMS ALL OTHER REPRESENTATIONS AND WARRANTIES, EXPRESSED OR IMPLIED, REGARDING THE PRODUCTS, INCLUDING ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. CEPHEID SHALL HAVE NO STRICT LIABILITY, PRODUCTS LIABILITY OR NEGLIGENCE, WHETHER ACTIVE OR PASSIVE.

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Table of ContentsPreface: Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Chapter 1: Theory of Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91.2 Heating/Cooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.3 Optical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.4 Reaction Tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121.5 Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Chapter 2: Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.1 Installing the SmartCycler® System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.2 Installing Additional SmartCycler Processing Blocks . . . . . . . . . . . . . . . . 192.3 Opening the SmartCycler Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .222.4 Closing the SmartCycler Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .232.5 Uninstalling or Reinstalling the SmartCycler Software . . . . . . . . . . . . . .23

Chapter 3: SmartCycler Software Overview . . . . . . . . . . . . . . . . . . . . . 253.1 Definitions of Terms Used in the SmartCycler Software . . . . . . . . . . . . .273.2 Background Subtraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .303.3 Cycle Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .353.4 A Quick Guide to Run Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

Chapter 4: The SmartCycler Software . . . . . . . . . . . . . . . . . . . . . . . . . . 514.1 Introduction to the SmartCycler Software . . . . . . . . . . . . . . . . . . . . . . . . .534.2 SmartCycler Software Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .554.3 The Create Run Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704.4 The View Results Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

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4.5 The Define Protocols Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1044.6 The Define Graphs Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1144.7 The Stop Run Button . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1234.8 The Check Status screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254.9 The Maintenance Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Chapter 5: Tutorial: Running SmartCycler Assays . . . . . . . . . . . . . . . 1295.1 Using the SmartCycler® Reaction Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . 1315.2 Running a B-Actin Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355.3 Applying Quantitative Analysis to the B-Actin Assay . . . . . . . . . . . . . .1445.4 Setting Up Melt Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1545.5 Overview: Running an Assay With an Internal Control . . . . . . . . . . . . . . 1585.6 Overview: Running a Multiplex Assay with a

Quantitative Internal Control (QIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .160

Chapter 6: Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1656.1 Removing and Replacing I-CORE® Modules . . . . . . . . . . . . . . . . . . . . . . . 1676.2 I-CORE Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1736.3 Disinfecting the SmartCycler System and I-CORE Modules . . . . . . . . . 1736.4 Disinfecting the Tube Rack and Cooling Block . . . . . . . . . . . . . . . . . . . . 174

Chapter 7: Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Appendix A: User Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197A.1 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197A.2 User Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

Appendix B: Optical Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203B.1 Optical Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203B.2 Optical Calibration Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204B.3 Optical Calibration Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209B.4 Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212B.5 Optical Calibration Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214B.6 Verification Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

Appendix C: Windows 2000 and Windows XP withthe SmartCycler Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

C.1 Logging on to Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220C.2 Performing Database Functions (Archiving and Retrieving) . . . . . . . 220C.3 Desktop Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220C.4 Firewalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

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Appendix D: SmartCycler Technical Specifications. . . . . . . . . . . . . . 223

Appendix E: SmartCycler Software Release Notes . . . . . . . . . . . . . . 225

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

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Preface: Safety Information

Preface: Safety InformationSymbols and Conventions Used in This ManualSignal words are as follows:

Danger — indicates an imminently hazardous situation, which, if not avoided, will result in death or serious injury (most extreme).

Gefahr— zeigt eine gefährliche Situation an, die, wenn nicht vermieden, Tod oder die ernste Verletzung ergibt (am extremsten).

Warning — indicates a potentially hazardous situation which, if not avoided, could result in serious injury or death.

Warnung — zeigt eine möglicherweise gefährliche Situation an, die, wenn Sie nicht vermieden, konnten ernste Verletzung oder Tod ergeben.

Caution — indicates a potentially hazardous situation which may result in minor or moderate injury to the user or damage to the equipment.

Vorsicht — zeigt eine möglicherweise gefährliche Situation an, die kann geringe oder gemäßigte Verletzung zum Benutzer oder zur Beschädigung der Ausrüstung ergeben.

Sample hazard symbols are shown below:Beispielgefahrsymbole werden gezeigt unter:

Warning: This heading and symbol are used to indicate that non-compliance with instructions or procedures may lead to injury or even death.

Warnung:Dieser Kopftext und Symbol werden benutzt, um anzuzeigen, daß Zuwiderhandlung gegen Anweisungen oder Prozeduren zu Verletzung oder sogar Tod führen kann.

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Caution: This heading and symbol are used to indicate that non-compliance with instructions or procedures may cause damage to the instrument.

Vorsicht: Dieser Kopftext und Symbol werden benutzt, um anzuzeigen, daß Zuwiderhandlung gegen Anweisungen oder Prozeduren Beschädigung des Instrumentes verursachen kann.

Note: This heading is used to bring your attention to topics of importance.

Safety LabelsThere are two safety labels on the SmartCycler instrument. The labels contain symbols that indicate the type of potential hazard, as well as signal words accompanied by definitions of the particular hazard.

Es gibt zwei Sicherheit Kennsätze auf dem SmartCycler-Instrument. Die Kennsätze enthalten Symbole, die die Art der möglichen Gefahr anzeigen, sowie die Kennzeichenwörter, die von den Definitionen der bestimmten Gefahr begleitet werden.

The safety label below is located inside the SmartCycler lid.

Dieser Sicherheit Kennsatz befindet sich innerhalb der Kappe des SmartCycler.

The safety label below is located on the back panel of the SmartCycler instrument.

Dieser Sicherheit Kennsatz ist auf der Rückplatte des SmartCycler.

DISCONNECT ALL ELECTRICAL CORDS FROM DEVICEBEFORE PROCEEDING FURTHER. CONSULT USERMANUAL FOR ADDITIONAL INSTRUCTIONS.

WARNING:

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SmartCycler Safety ProceduresBefore using the SmartCycler system, read the following safety information. Ensure that anyone using the instrument has been instructed in general lab safety practices and the specific safety practices for the instrument.

Bevor Sie das SmartCycler-System verwenden, lesen Sie die folgenden Sicherheit Informationen. Stellen Sie sicher, daß jedermann, welches das Instrument verwendet, in der allgemeinen Laborsicherheit Praxis und in der Besonderesicherheit Praxis für das Instrument angewiesen worden ist.

Moving the Instrument

The SmartCycler weighs 22 pounds (10 kg). Safety training for proper lifting techniques is recommended. Improper lifting can cause painful and perhaps permanent back injury.

Das SmartCycler wiegt 10 Kilogram. Sicherheit Training für korrekte anhebende Techniken wird empfohlen. Das unsachgemäße Anheben kann schmerzliche oder permanente rückseitige Verletzung verursachen.

Smart CyclerMAXIMUM CHAIN OF 3 UNITS

ONLY

¤USC

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General Laboratory SafetyAllgemeine LaborSicherheit

Your laboratory should have all equipment ordinarily required for the safety of individuals working with chemicals. A partial list includes fire extinguishers, first-aid equipment, safety shower and eye-wash fountain, and spill-cleanup equipment.

Ihr Labor sollte alle Ausrüstung haben, die gewöhnlich für die Sicherheit der Einzelpersonen angefordert wird, die mit Chemikalien arbeiten. Eine teilweise Liste schließt Feuerlöscher, Erste-Hilfeausrüstung, Sicherheit Dusche mit Auge-Wäschebrunnen, und Streuungreinigungausrüstung.

ClassificationKlassifikation

The SmartCycler instrument is classified as:

Das SmartCycler-Instrument wird wie eingestuft:

ISM instrument (Industrial Scientific Medical Device), medium sized, for indus-trial, laboratory and domestic use.

ISM-Instrument (industrielle wissenschaftliche medizinische Einheit), mittelgroß, für industrielles, Labor und inländischen Gebrauch.

Designed for stationary operation.

Entworfen für stationäre Operation.

Intended for worldwide use.

Beabsichtigt für weltweiten Gebrauch.

Intended for evaluating preprocessed biological material.

Beabsichtigt für vorbearbeitetes biologisches Material Auswertens.

Note on Use with Infectious MaterialAnmerkung über Gebrauch mit Ansteckendem Material

The instrument may not be used to analyze infectious materials, unless additional safety measures to ensure safe sample handling (e.g., placing the instrument in a laminar flow biological safety cabinet) are taken beforehand.

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Das Instrument darf nicht benutzt werden, um ansteckende Materialien zu analysieren, es sei denn zusätzliche Sicherheitsmaßnahmen die sichere Probe-behandlung sicherzustellen (z.B., das Instrument in einen blätterigen Luftstrom biologischen-Sicherheit Schrank legend), vorher genommen werden.

Marks of ConformityMarkierungen der Übereinstimmung

The SmartCycler instrument has been manufactured according to EN 61010-1 (Safety Regulations for Measuring, Control, and Laboratory Instruments); Part 1: General Requirements [IEC 1010-1 + A1: 1992, modified]). The SmartCycler has been checked in accordance with all relevant safety standards before leaving the factory.

Das SmartCycler-Instrument ist hergestellt worden entsprechend en 61010-1 (Sicherheit Regelungen für Messen, Steuer- und Laborinstrumente); Teil 1: Allgemeine Anforderungen [IEC 1010-1 + A1: 1992, geändert]. Das SmartCycler ist in Übereinstimmung mit allen relevanten Sicherheit Standards überprüft worden, bevor man die Fabrik verließ.

Electrical SymbolsElektrische Symbole

The symbols shown below are affixed to the SmartCycler. Read the explana-tions and make sure you understand what the symbols mean before you interact with the instrument in any way.

Die Symbole, die auf dieser Seite gezeigt werden, werden zum SmartCycler hinzugefügt. Lesen Sie die Erklärungen und verstehen was die Symbole bedeuten bevor Sie auf das Instrument in jeder Hinsicht einwirken.

This symbol indicates the on position of the main power switch.

Dieses Symbol zeigt die Arbeitsstellung des Betriebsschalters an.

This symbol indicates the off position of the main power switch.

Dieses Symbol zeigt die Ausschaltstellung des Betriebsschalters an.

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This symbol indicates that a protective grounding terminal must be connected to earth ground before any other electrical connections are made to the instrument.

Dieses Symbol zeigt an, daß ein Schutzleiteranschluß an Masse Boden angeschlossen werden muß, bevor alle anderen elektrischen Bezie-hungen zum Instrument hergestellt werden.

This symbol indicates that this terminal either receives or delivers alternating current or voltage.

Dieses Symbol zeigt an, daß dieses Terminal entweder empfängt oder liefert Wechselstrom oder Spannung.

Contact InformationCepheid904 Caribbean DriveSunnyvale, CA 94089-1189

Tel: (408) 541-4191Fax: (408) 541-4192Technical Support toll-free: (888) 838-3222

(5:00 am to 5:00 pm, Pacific time, Monday–Friday)Email: [email protected]

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Chapter 1: Theory of Operation

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.2 Heating/Cooling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.3 Optical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.4 Reaction Tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.5 Calibration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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Introduction

1.1 IntroductionThe Cepheid SmartCycler System is an integrated DNA/RNA amplification and detection instrument system based on the proprietary microprocessor-controlled I-CORE® (Intelligent Cooling/Heating Optical Reaction) module. Ease of use is designed into the system through the SmartCycler Software. Each SmartCycler processing block contains sixteen independently controlled, programmable I-CORE modules, each with one reaction site. Thermally optimized proprietary reaction tubes combined with the unique design of the I-CORE modules allow very rapid cycling and faster amplification and detection. Up to six SmartCycler processing blocks can be daisy-chained together, allowing simultaneous custom analysis of 96 discrete samples.

The SmartCycler System is ideally suited to research, such as PCR and RT-PCR, that requires automatic, repeated, rapid heating and cooling cycles for test samples. Specific sequences can be detected using hybridization probes or intercalating dyes (except Ethidium bromide). The system has the capacity to store any number of user-generated protocols, limited only by available disk space. All data, including cycling programs and assay results, are stored in a database. Selected data can be exported to spreadsheet programs such as Microsoft Excel.

Figure 1-1: Schematic diagram of the major components of an I-CORE module.

I-CORE

Reaction tube

Fan Emission detectionoptical block

Excitationoptical block

Notch

Heater

circuit board

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1.2 Heating/CoolingThe disposable reaction tube is inserted into the I-CORE thermal cycling module for amplification. The chamber consists of two heater plates made of a ceramic material that has high thermal conductivity to assure temperature uniformity and rapid heat transfer. Resistive heater elements are deposited on the ceramic plates using thick film technologies and a thermistor attached directly to each plate monitors its temperature. Cooling is accomplished with a high-efficiency fan that moves ambient air across the heater plates. The thermal cycling chamber’s temperature is controlled by the instrument’s firmware. The firmware incorporates a control loop to ensure rapid heating of the plates and to control their temperature overshoot around the desired point, allowing the temperature of the fluid in the reaction tube to be changed rapidly and precisely.

1.3 Optical SystemThe SmartCycler optical system uses high intensity light-emitting diodes (LEDs), silicon photodetectors, and appropriate filters for excitation and detection of four different spectral bands. The optical system includes two optical blocks: (1) a four-color excitor module and (2) a four-color detector module. These blocks are positioned within the device such that their apertures mate with the optical windows of the reaction tube, allowing excitation and emission detection of the reaction mixture. Figure 1-2 shows the mechanical layout of the optical blocks.

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Optical System

Figure 1-2: Schematic diagram of the SmartCycler optical blocks.

The excitation and detection spectral bands for the four channels are:

Excitation Optical Block

Reaction tube

LED Filters Lens Mirror

Lens

Emission Detection Optical Block

Reaction tube

Mirror

Lens

Filters Detector

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Table 1–1: Excitation and detection ranges for SmartCycler optical channels.

Although the SmartCycler System is calibrated using the five dyes mentioned in the table above, other dyes with excitation and emission wavelengths within these ranges can be used if user-defined optical calibration is performed.

By using probes labeled with different fluorescent dyes, up to four dyes and targets can be amplified and detected simultaneously in a single reaction mixture. The optical system always collects data from all four channels. Because the emission spectra of fluorescent dyes can overlap, the presence of a particular dye will result in signals in more than one of the channels. Appro-priate calibration and data analysis algorithms are used to separate the signal from each of the dyes.

1.4 Reaction TubeThe unique SmartCycler polypropylene reaction tube (Figure 1-3) is designed for optimal thermal and optical characteristics. The thin reaction tube allows rapid rates of heating and cooling of the reaction mixture, and hence rapid amplification. The reaction tube also includes two optical detection windows at a 90° angle to each other along the bottom edges of the tube. These optical windows interface with the I-CORE optical blocks to allow fluorescence excitation and emission detection to occur.

Optical Channel

Excitation (nm)

Emission (nm)

Calibrated Dye(s)

Supported Quenchers

1 450–495 510–527 FAM BHQ, Eclipse, DABCYL

2 500–550 565–590 Cy3™/TETAlexa Fluor 532

BHQ, Eclipse, DABCYL

3 565–590 606–650 Texas Red™ BHQ, Eclipse, DABCYL

4 630–650 670–750 Cy5™Alexa Fluor 647

BHQ, Eclipse

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Calibration

Figure 1-3: SmartCycler 25 μL reaction tube.

1.5 CalibrationThe thermistors used to monitor the reaction chamber temperature are calibrated to ±0.50 °C using National Institute of Standards and Technology (NIST)-traceable standards. During the manufacturing process, the temper-ature of the heating system is measured at two temperatures, 60 °C and 95 °C. Calibration coefficients which correct for small errors in the raw thermistor readings of the heaters are stored in the memory of each I-CORE module.

The optical system is calibrated using standard concentrations of the individual unquenched dye-oligos to be detected. For each optical channel, the signal produced by buffer alone (the blank signal) is subtracted from the raw signal produced by the dye-oligo standard to determine the spectral characteristics. Using the individual spectral characteristics of the pure dye-oligos, signals from an unknown mixture of dye-oligos can be resolved into corrected signals for the individual dye-oligos in the mixture.

Opticaldetectionwindow

Opticalexcitationwindow

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14 D1819 Rev D

Chapter 2: Installation

2.1 Installing the SmartCycler® System. . . . . . . . . . . . . . . . . . . . . . . . 17

2.2 Installing Additional SmartCycler Processing Blocks . . . . . . . . . 19

2.3 Opening the SmartCycler Software . . . . . . . . . . . . . . . . . . . . . . . . 22

2.4 Closing the SmartCycler Software . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.5 Uninstalling or Reinstalling the SmartCycler Software . . . . . . . 23

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Installing the SmartCycler® System

2.1 Installing the SmartCycler® System

Setting Up the SmartCycler Computer

Note: Windows requires all users to logon at startup. For your convenience, a user with administrative rights has been set up at the factory and will automati-cally log on at startup. For Windows 2000 the user name is Administrator and the password field is left blank. For Windows XP the user name is Cepheid and the password field is left blank.

If new users are created, all power settings must be set to Never for each new user. Data will be lost if the system turns off or goes into hiber-nation during a run. Windows 2000: select Start > Settings > Control Panel > Power Options. Set Power Schemes to Always On and verify that Turn Off Monitor, Turn off hard disks, and System Standby are set to Never. Windows XP: select Control Panel > Performance and Mainte-nance > Power Options. Verify that Turn Off Monitor, Turn off hard disks, and System Standby are set to Never. For laptop computers, make sure that Plugged in and Running on batteries are set to Never.

Do not install additional equipment by a USB connection (e.g., USB hub or Zip® drive, etc.).

Do not install any other software programs on the SmartCycler computer. Please call Cepheid Technical support at (888) 838-3222 before installing any Cepheid software programs.

1. Set up the computer as directed in the manufacturer's instructions.

2. Turn on the computer.

3. Confirm that the SmartCycler Software icon is on the computer desktop (Figure 2-1). If the icon is present, the software has been installed successfully.

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Figure 2-1: The SmartCycler Software icon.

Setting up the SmartCycler Processing Block1. Unpack the SmartCycler processing block. The following should be

included:

1 SmartCycler processing block1 power cord1 USB cable

2. Set up the SmartCycler system on a level, hard surface. Multiple processing blocks should be placed at least 2 inches apart for efficient cooling. Do not cover or obstruct air vent holes on the sides of the processing block(s). Pro-cessing blocks can be placed adjacent to each other but be careful not to block the air flow between them (for example, with folders or papers).

3. Connect the power cord to the SmartCycler processing block and into a surge protector power strip or into a power outlet.

4. Insert the flat end of the USB cable into the connector labelled with the USB icon on the back of the computer as shown in Figure 2-2. Insert the square end of the USB cable into the square USB connector on the back panel of the SmartCycler processing block.

Figure 2-2: Connecting the first SmartCycler processing block to the computer via a USB cable.

USB USB

USB

Computer

To ACoutlet

To ACoutlet

To

To mouse

SmartCyclerprocessing block

USB cablemonitor

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Installing Additional SmartCycler Processing Blocks

5. Turn on the SmartCycler processing block.

6. Double-click on the SmartCycler icon on the Windows desktop (Figure 2-1) or, from the Windows Start menu select Programs > Cepheid > SmartCycler.

7. The SmartCycler splash screen will appear, and two identifying letters should light up and momentarily flash in the small round display areas on the left side of the top of the SmartCycler processing blocks. The flashing indi-cates that the firmware is downloading from the computer to the process-ing block. After a short time, the View Results screen will be displayed.

8. To shut down the SmartCycler Software, choose Exit from the File menu, or click the close box in the top right corner of the SmartCycler Software win-dow.

2.2 Installing Additional SmartCycler Processing Blocks

1. Unpack the additional SmartCycler processing blocks, power cords and USB cables. Set up the SmartCycler processing blocks on a level hard surface at least 2 inches apart for efficient cooling. Do not cover or obstruct air vent holes on the sides of the processing blocks. Processing blocks can be placed adjacent to each other but be careful not to block air flow between them (for example, with folders or papers).

2. Close all software programs, including the SmartCycler Software.

3. Turn the previously installed SmartCycler processing block’s power off.

Note: Do not turn off or disconnect the processing block(s) while the Smart-Cycler Software is open.

4. Connect the power cord to the second SmartCycler processing block and into a surge protector power strip or into a power outlet. (Figure 2-3).

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Figure 2-3: Connect the power cord to the second SmartCycler processing block and into a power outlet.

5. Insert the flat end of the USB cable into the second connector labelled with the USB icon as shown in Figure 2-4. Insert the square end of the USB cable into the square USB connector on the back panel of the second SmartCycler processing block.

Figure 2-4: Connecting the second processing block to the computer via a USB cable.

6. If daisy-chaining three or four processing blocks, chain the first two blocks to the two USB ports on the computer, then connect the third and fourth processing blocks to the first and second processing blocks (Figure 2-5).

Warning:Do not connect more than three SmartCycler processing blocks in a single daisy-chain with USB cables.

USB USB USB USB

USB

Computer

First SmartCycler

To ACoutlet

To monitor

To mouse

Second SmartCyclerprocessing block (1) processing block (2)

USB cable

To AC outletTo AC outlet

USB USB USB USB

USB

To ACoutlet

To

To mouse

Computer

USB cable

First SmartCycler Second SmartCyclerprocessing block (1) processing block (2)

USB cable

To AC outlet

monitor

To AC outlet

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Installing Additional SmartCycler Processing Blocks

Figure 2-5: Daisy-chaining four SmartCycler processing blocks.

Figure 2-6: Daisy-chaining six SmartCycler processing blocks.

USB

Computer

To ACoutlet

To

USB cable USB cable

1 3

2

USB cable

4

To AC outlet

USB cable

To

To ACoutlet

To ACoutletmouse

monitor

To AC outlet

USB

Computer

To ACoutlet

To ACoutlet

To

To

USB cable

USB cable

USB cable USB cablemonitor

1 3 5

2 4 6

To ACoutlet

To ACoutlet

USB cableUSB cable

To ACoutlet

To ACoutlet

To ACoutlet

mouse

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7. Turn the power on to all of the SmartCycler processing blocks in the order they are connected to the computer (i.e., processing block 1 first, then pro-cessing block 2, etc.; refer to Figure 2-6).

Important: Turning off the power to one processing block will cause all processing blocks ‘downstream’ to lose their connection to the computer.

8. Windows XP only (Windows 2000 users skip to step 9): if the Found New Hardware Wizard window opens, click Next to continue. The Hardware Installation window will open. Click Continue Anyway, then click Finish to close the wizard.

9. Open the SmartCycler Software. The splash screen should appear, and two “A’s” should light up and flash on the top of the SmartCycler processing block (if more than one processing block is installed, the first will be lettered “A”, the second “B” and so on). This indicates that the firmware has success-fully downloaded from the computer to the processing block.

2.3 Opening the SmartCycler SoftwareTo open the SmartCycler Software:

1. Turn on the SmartCycler processing block; 0 and 1 will appear on the top of the processing block.

2. Double-click the SmartCycler icon on the Windows desktop, or select Start > Programs > Cepheid > SmartCycler.


3. The SmartCycler splash screen will appear and the 0 and 1 on the top of the processing block will change to letters. The flashing indicates that the firm-ware is downloading from the computer to the processing block. The first processing block will have two A’s, the second block will have two B’s, etc.

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Closing the SmartCycler Software

4. Once the firmware is downloaded and the software is open, the View Results screen will be displayed.

The calibration status of all I-CORE modules in each processing block are checked when the software is opened. If any module has been started 900 or more times since the last calibration, the software will display a warning that calibration is needed. See section 4.9, “The Maintenance Screen” on page 126 for more information on checking the calibration status of I-CORE modules.

2.4 Closing the SmartCycler SoftwareTo close the SmartCycler Software, click the close box in the top right corner of the SmartCycler Software window or select Exit from the File menu.

Note: Do not turn off or disconnect the processing block(s) while the Smart-Cycler Software application is open.Close the SmartCycler Software before connecting a SmartCycler processing block or changing the order in which the processing blocks are connected to the system, otherwise the software will not function correctly.

2.5 Uninstalling or Reinstalling the SmartCycler Software

The SmartCycler Software has been pre-installed on the system’s computer. If the software becomes corrupted, or if a major system failure occurs, do not attempt to re-install the software. Please call Technical Support for assistance, to minimize the chance of permanent data loss.

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Chapter 3: SmartCycler Software Overview

3.1 Definitions of Terms Used in the SmartCycler Software . . . . . . 27

3.2 Background Subtraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3.3 Cycle Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.4 A Quick Guide to Run Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

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26 D1819 Rev D

Definitions of Terms Used in the SmartCycler Software

3.1 Definitions of Terms Used in the SmartCycler Software

Alexa Fluor 532 — Alexa 532 fluorescent dye, absorbance peak at 532 nm, emission peak at 554 nm.

Alexa Fluor 647 — Alexa 647 fluorescent dye, absorbance peak at 650 nm, emission peak at 668 nm.

Assay Channel — a channel whose Usage has been set to Assay.

Background — initial fluorescent signal derived from unbound probe, non-specific cleavage of probe or sample auto-fluorescence; background signal is not produced by amplified product.

Background Subtraction — a method that adjusts the displayed signal for all samples to the common starting point of zero. The SmartCycler Software calculates and subtracts the average background fluorescence individually for each sample. See section 3.2 “Background Subtraction” on page 29 for a detailed discussion.

Boxcar Average — a statistical technique for reducing noise and smoothing a growth curve. For each cycle, the fluorescent signal is measured and averaged with a user-defined number of previous cycles minus one. For example, if 3 is entered into the Boxcar Average column, the fluorescent signal of the current cycle and the fluorescence values of the two previous cycles are averaged and displayed.

Channel — there are four optical channels in each I-CORE module. Each channel illuminates the reaction mixture with a particular spectral band of LED light, and detects emissions from the reaction mixture within a particular spectral band. The excitation and emission spectral ranges are specified by the dye set and the channels are calibrated to those ranges.

Control Channel — a channel whose Usage has been set to Internal Control or QIC (Quantitative Internal Control).

Cy3™ — Carbocyanine Cy3 fluorescent dye, absorbance peak at 550 nm, emission peak at 564 nm.

Cy5™ — Carbocyanine Cy5 fluorescent dye, absorbance peak at 649 nm, emission peak at 670 nm.

Cycle Threshold (Ct) — the first cycle in which there is a significant increase in fluorescence above the background or a specified threshold. The Ct can be

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determined by analyzing the growth curve (Primary Curve) or the second deriv-ative of the growth curve (2nd Deriv).

Dye Set — the dye set specifies the calibration data used for optical detection of dyes in the four channels. The four factory calibrated dye sets are FCTC25 (FAM, Cy3, Texas Red, Cy5, 25 μL tubes), intcltr_dye (25 μL tubes), FTTC25 (FAM, TET, Texas Red, Cy5, 25 μL tubes) and FATA25 (FAM, Alexa Fluor 532, Texas Red, Alexa Fluor 647, 25 μL tubes).

FAM — Carboxyfluorescein fluorescent dye, absorbance peak at 494 nm, emission peak at 518 nm.

Growth Curve — a plot of fluorescence vs. cycle number. A real time growth curve should have three distinct phases: baseline, log-linear and plateau. The increase in fluorescence is proportional to the amount of amplicon generated and can be used to define the cycle threshold.

Internal Control — a channel designated as Internal in the Usage column of the Analysis Settings. This channel is monitored for a threshold crossing within a specified range of cycles, and validates or invalidates the results for that site. Data is not normalized to this internal control.

Melt Curve — a plot of fluorescence vs. temperature. When a melt is performed with an intercalating dye (except Ethidium bromide), analysis of the melt curve can identify the Tm (melting temperature) of the amplified product (see the definition of Tm, below).

Protocol — the thermal conditions for a run. The protocol also defines the step at which optical data is collected. A different protocol can be applied to each site or group of sites in a run.

Quantitative Internal Control (QIC) — when a channel is designated as QIC, the cycle threshold values of all channels designated as Assay are normalized to the cycle threshold of the QIC. This channel is monitored for a threshold crossing within a specified range of cycles, and validates or invalidates the results for that site.

Run — the execution of one or more selected protocols, performed simulta-neously on a group of sites. A run includes all the setup parameters, plus the acquired data and analysis. The setup parameters include the run name, notes, dye set, selected sites and protocol(s).

2nd Derivative Curve — the 2nd derivative of the growth curve (primary curve) is the rate of change of the growth curve slope. The highest peak of the 2nd derivative curve represents the point of maximum curvature of the growth

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Background Subtraction

curve or the transition from background fluorescence to amplified product fluorescence. Analysis of the 2nd derivative curve can be used to define the cycle threshold.

Site — individual thermal cycling module (I-CORE) used for sample testing. There are 16 sites in a SmartCycler processing block.

Stage — a subset of a protocol. A set of time/temperature steps (for example, holding the site at a certain temperature, or cycling it repeatedly through a series of temperature changes). A stage can also be a melt curve, a slow temperature ramp combined with continuous optical data collection.

Standard — a sample of known concentration used to quantitate unknowns.

Step — a subset of a protocol stage. A step defines the temperature and time to hold a site.

Temperature Ramp — the rate at which the temperature increases or decreases between programmed temperature steps. This can be a user-defined ramp for amplification or melt curve functions.

TET — Tetrachloro-6-carboxyfluorescein fluorescent dye, absorbance peak at 522 nm, emission peak at 538 nm.

Texas Red (TxR) — Sulforhodamine 101 fluorescent dye, absorbance peak at 598 nm, emission peak at 615.5 nm.

Threshold — a user-defined fluorescence value used to evaluate the optical signal. If the primary curve crosses the threshold or if the 2nd derivative peak is above the threshold, it is counted as a positive result. If the primary curve or 2nd derivative peak remains below the threshold, it is counted as a negative result.

Tm — the melting temperature of a specific DNA sequence, which is a function of the number of base pairs and % GC content. The Tm is defined as the temperature at which 50% of the DNA is double-stranded and 50% is single-stranded. The Tm can be empirically derived by performing a melt curve and plotting the absolute value of the first derivative of the melt curve against temperature. The highest peak of this curve corresponds to the melting temperature.

3.2 Background SubtractionBackground fluorescence is a property of real time PCR and can be defined as the fluorescence derived from unbound probe, free dye, non-specific cleavage

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of probe or sample auto-fluorescence. Background fluorescence represents the baseline phase of a real time PCR growth curve. The log-linear phase represents both background fluorescence and fluorescence corresponding to amplified DNA. The growth curve plateaus when critical components of the PCR reaction become rate-limiting. After background fluorescence is subtracted from the raw signal, the net fluorescence corresponds only to amplified DNA. Therefore, the background must be subtracted out for qualitative and quantitative analysis of real time PCR data. The two graphs below demonstrate the effect of background subtraction and drift correction on a growth curve. The initial fluorescence is 80 fluorescent units with background subtraction OFF and displays slight upward drift. When background subtraction is applied (ON) the background fluorescence is corrected to zero and the background drift is elimi-nated. Background subtraction must be ON to apply qualitative or quantitative analysis to the data and achieve accurate cycle threshold analysis. To change the background subtraction setting, click Analysis Settings under the Views list and click in the Bkgnd Sub column. Select ON or OFF from the drop-down menu and click Update Analysis to implement the change.

Figure 3-1: Effect of background subtraction on a growth curve.

The SmartCycler Software calculates the background subtraction in two steps. The first step corrects for any drift (positive or negative slope) in the background signal. The second calculates the average background signal and subtracts that signal from each data point so that the resultant growth curve represents amplified product only.

The software Bkgnd Min and Max Cycle defaults of cycle 5 and cycle 40 are used in the following figures to illustrate how background subtraction is imple-

Background subtraction OFF Background subtraction ON

~80 fluorescent units Zero fluorescence

Level baseline

Log-linear phase

Plateau

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mented in the SmartCycler Software. Cycles 5 to 40 define the range that can be used to calculate the average background fluorescence and slope of the background fluorescence. At least five data points (cycles) are required for this calculation. To avoid using fluorescence data derived from amplified DNA, the four most recent cycles of data are not used in the background subtraction calculation. Initially, the background subtraction is not applied until cycle 13 if the Bkgnd Min Cycle is defined as cycle 5. Five cycles of data (5,6,7,8,9) plus the four most recent cycles (10,11,12,13) are required before the first calculation.

Figure 3-2: The magnitude of the background fluorescence for this sample is 200 fluores-cence units. Raw fluorescence is displayed until background subtraction is applied at cycle 13.

Figure 3-3: Background subtraction is first applied at cycle 13.

Cycle 5; background subtraction and drift correction are not applied yet.

Cycle 13; background subtraction and drift correction are applied when cycle 13 is completed.

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After the optical read in each cycle, the average background fluorescence and slope are recalculated and subtracted from the measured fluorescence. For example, after the optical read for cycle 14, background subtraction and drift correction are recalculated and applied to the data for cycles 1 through 14. This repetitive process continues until a threshold crossing occurs.

Note: When a cycle threshold is detected, there is no further background subtraction recalculation and the same value of background fluores-cence and slope correction are subtracted for the remainder of the run.

All calculations are performed and applied individually for each site. This method ensures that each sample, regardless of when the cycle threshold occurs, has the benefit of using the maximum background data to calculate the most accurate background subtraction and drift correction.

Figure 3-4: Background subtraction calculation stops when a cycle threshold is detected.

If cycle threshold is not detected the subtraction and drift correction will continue until the background max cycle is reached.

If a growth curve displays significant negative drift it is possible that the threshold needs to be lowered or the background min and max cycles need to be adjusted. The example below in Figure 3-5 demonstrates the effects of a threshold that is set too high. If the threshold is set too high a threshold crossing will not occur and the background subtraction and drift correction calculation will continue until the background max cycle is reached. A negative

Cycle threshold at cycle 32.39.

Background subtraction and drift correction stop.

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slope in the baseline occurs because the positive slope of the growth curve is included in the background subtraction and drift correction calculation.

Figure 3-5: An example of a growth curve if the threshold is set too high.

It is possible to check if the negative slope in the baseline is a result of the background subtraction and drift correction calculation by turning the background subtraction OFF. When the background subtraction is turned OFF for the growth curve in Figure 3-5 the baseline is linear and the curve is a classical S-shape. Therefore, it is evident that the threshold needs to be set lower. If the growth curve appears to enter the log linear phase before cycle 13, decrease the background minimum cycle number.

Appearance of negative drift be-cause positive slope of the growth curve was included in the background subtraction and drift correction calculation.

Background subtraction ONThreshold set too high.

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Figure 3-6: An example of a growth curve with background subtraction OFF.

Raw fluorescence data provides essential information about the magnitude of the background signal and the shape of the growth curve without drift correction.

If the threshold is set lower and a Ct is detected the shape of the growth curve with the background subtraction turned ON and OFF should be similar. In Figure 3-7 the threshold is set appropriately (above the baseline and at the beginning of the log-linear phase) and a threshold crossing occurred at 31.28 cycles. Therefore, the re-calculation of the background subtraction and drift correction calculation stopped approximately 4 cycles before the threshold crossing and the baseline is adjusted appropriately.

Baseline is linear with back-ground subtraction OFF.

Background subtraction OFF

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Cycle Threshold

Figure 3-7: Example demonstrating appropriate threshold placement and background subtraction calculation.

3.3 Cycle ThresholdReal time PCR offers the ability to analyze data during the log-linear phase rather than the end point of a reaction. Real time PCR either incorporates a non-sequence specific intercalating dye (note: Ethidium bromide cannot be used as an intercalating dye on the SmartCycler system) or a fluorescent dye-labeled sequence-specific probe, such as TaqMan®, to visualize and monitor the amplified product in real time. A plot of the fluorescence vs. cycle number produces a sigmoidal shaped curve called a growth curve. To analyze real time PCR assays, the log-linear phase of the reaction is used to determine the cycle threshold (Ct) for each sample. The most basic definition of a Ct is the first cycle in which there is a significant increase in fluorescence above the background fluorescence. In practical use, the Ct can be determined by more than one method to provide an accurate comparison of PCR amplified samples. Real time PCR assays can be developed and performed to provide reproducible qualitative and quantitative analysis of DNA and RNA.

The SmartCycler Software provides multiple methods for determining the Ct. All data analysis options are selected in Analysis Settings in the Create Run or View Results screen. If Primary Curve is selected in the Curve Analysis column, the Ct is detected and reported at the cycle where the primary curve crosses the threshold. Moving the threshold up or down will affect when the growth curve intersects the threshold. Thus, changing the threshold will change the Ct. The threshold can be set very close to the background fluorescence to achieve a faster answer or a more sensitive detection limit. However, if the threshold is

Threshold crossing at cycle 31.28.

Background subtraction and drift correction stopped.

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set too close to background fluorescence, background noise could cross the threshold and be reported incorrectly as the Ct. The threshold can be set at a higher fluorescence which would avoid false Cts, but could reduce sensitivity.

Figure 3-8: Primary Curve analysis.

The 2nd Deriv (second derivative) can also be selected for the Curve Analysis. The 2nd derivative represents the rate of change in the slope of the growth curve. The highest peak of the 2nd derivative represents the point of maximum curvature of the growth curve. The threshold defines only a minimum peak height for determining Ct. If the 2nd derivative peak is above the threshold, a Ct is reported. If the peak is below the threshold, no Ct is reported. The threshold can be set significantly above the background fluorescence without affecting the Ct. 2nd derivative analysis can potentially be more reproducible from run to run because the Ct is determined by the shape of the growth curve and is independent of the magnitude of the fluorescent signal.

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Cycle Threshold

Figure 3-9: 2nd derivative Curve Analysis.

To define a threshold, select Manual, Run-based Auto or Site-based Auto from the Thresh Setting column drop-down menu. If Manual is selected, the threshold line will be displayed on the graph at the specific fluorescence value entered in the Manual Thresh Fluor Units column. If the optics graph is set to show the horizontal threshold line, it will be shown in red if the Curve Analysis setting is Primary Curve and blue if the Curve Analysis setting is 2nd Deriv-ative. If the default of 30 is accepted, the threshold line is set at 30 fluorescent units on the Y-axis.

If Run-based Auto or Site-based Auto is selected, the threshold is defined as a function of the background fluorescence noise by entering the number of standard deviations (SDs) above background fluorescence in the Auto Thresh #SD’s column. The Auto Min Cycle (default = cycle 5) and Auto Max Cycle (default = cycle 10) columns define the range of cycles used to calculate the standard deviation. The SmartCycler Software calculates the standard deviation for the designated cycles of each site in the run. For Run-based Auto, the largest standard deviation is multiplied by the number entered in Auto Thresh #SD’s to define the threshold. For Site-based Auto, the standard deviation for each site is multiplied by the number entered in Auto Thresh #SD’s to define the threshold for that site.

See section 4.4, “The View Results Screen” on page 76 for details describing how to implement data analysis options.

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3.4 A Quick Guide to Run Setup To start a run on the SmartCycler, you define a protocol in the Define Protocols screen and enter run information in the Create Run screen. Once the run has started the software will automatically switch to the View Results screen, in which the data can be viewed and analyzed. The instructions below are only an overview. For detailed information about each screen see Chapter 4 “The SmartCycler Software”.

Defining a ProtocolIf the protocol has already been defined skip to “Creating a Run” on page 39.

Figure 3-10: The Define Protocols screen.

1

2

3

4

5

1

1. Click the Define Protocols icon.

2. Click the New Protocol button.

3. Enter a unique Protocol Name.

4. Enter the thermal cycling parame-ters. See section 4.5, “The Define Protocols Screen” on page 104 for more details.

5. Click the Save Protocol button.

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A Quick Guide to Run Setup

Note: Only one optical read can be programmed in each stage and the optical read step must be a minimum of 6 seconds.

Creating a Run

Figure 3-11: The Create Run screen.

2

3

4

5

6

7

9

10

1

1. Click the Create Run icon.

2. Enter a Run Name.

3. Select a Dye Set.

4. Click the Add/Remove Sites button.

5. Highlight a protocol name.

6. Highlight site(s).

7. Click the right arrow.

8. Repeat steps 5–7 until all proto-cols and sites have been selected.

9. Click OK.

10. Click the Start Run button.

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Note: If a protocol is not listed in the Protocols dialog box, define the protocol in the Define Protocols screen, see Figure 3-10 or “Defining a Thermal Cycling Protocol” on page 106 for complete details.

After the run has been started, the software will automatically switch to the View Results screen. The user can monitor the temperature and optical data in real time, as well as select graphs, Analysis Settings, and Sample Type infor-mation while the run is in progress.

Note: Changes can be made to the Analysis Settings, graphs and Results Table before, during and after the run is complete unless using Advance To Next Stage. Previous runs can be viewed while runs are in progress.

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Viewing Results

Figure 3-12: The View Results screen.

Views list options include:

• Results Table — select to define the Sample Type (standard, unknown, or unused), enter standard concentrations, enter sample IDs, and display run results for each site.

• Analysis Settings — select to define how the data from each dye channel will be analyzed and to see the data analysis parameters (see “Selecting Analysis Settings” on page 78 for detailed information).

• Protocols — select to display the protocol(s) used in the selected run.

• Optical data (e.g., FAM) — displays a plot of fluorescence vs. cycle num-ber in real time for the specified channels (see section 4.6, “The Define Graphs Screen” on page 114 for detailed information).

• Temperature — displays temperature data in real time.

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• Standard Curve — displays a plot of log concentration vs. cycle threshold value for standards and unknowns. This graph will be blank unless stan-dards (STD) are included in the run.

• Melt — displays a plot of fluorescence vs. temperature in real time. This graph will be blank unless a melt curve stage was included in the protocol.

Viewing Temperature ProfilesTo view the temperature in real time click on the Temperature graph in the Views list.

Figure 3-13: A Temperature graph.

Notes: Click a Site ID to view an individual site. Select multiple sites by using the [Shift] key for contiguous sites and [Ctrl] for non-contiguous sites. Use [Ctrl] + [A] with the cursor on a Site ID to return to viewing all sites.

Right click on the graph for a menu of graph-related functions.

Viewing Optical DataTo view optical data, click on the appropriate graph name in the Views list. The example below (Figure 3-14) shows optical data for Channel 1 signal.

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Figure 3-14: Display of an optical graph (FAM).

• View growth curves with or without threshold or threshold crossing lines. See section 4.6, “The Define Graphs Screen” on page 114.

• View Primary curves, 2nd Derivative curves or both at the same time. See section 4.6, “The Define Graphs Screen” on page 114.

Notes: Make sure that the graph definition and Analysis Settings match. For example, if 2nd Derivative is selected in the Curve Analysis column of the Analysis Settings table, make sure that the 2nd Derivative curve is displayed on the graph.

Click a Site ID in the graph legend to view an individual site. Select multiple sites by using the [Shift] key for contiguous sites and [Ctrl] for non-contiguous sites. Use [Ctrl] + [A] with the cursor on a Site ID to return to viewing all sites.

The functions of the buttons located across the bottom of the View Results screen are as follows:

• Save Run — saves any changes made to the selected run including the Analysis Settings, Results Table, associated graphs and notes. The raw data is automatically saved at the end of the run.

• Export — opens the Export Data dialog box, to export optical, melt and temperature data, or the Results Table and Analysis Settings. See “Exporting Data” on page 96 for more information on exporting runs.

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• Report — opens the Report Preview, to view or print a report. See “View-ing and Printing Reports” on page 99 for more information on printing reports.

• Select Graphs — opens the Select Graphs dialog box, which lists all saved graphs that can be associated with the selected run. The default graphs are always associated with every new run. All graphs associated with the run will be displayed in the Views lists. See “Displaying Graphs” on page 90 for information on using the Select Graphs dialog box.

• View Another Run — opens the Select a Run dialog box, which lists all runs stored in the database that can be viewed. The runs are listed by name and date, in reverse chronological order (i.e., most recent first). The runs can be sorted by name, status, user or date by clicking on the col-umn header.

• Delete Run(s) — opens a dialog box to select one or more runs to delete.

• Update Analysis — implements changes made to the Results Table or Analysis Settings.

• Import Std Curve — opens the Select a Run dialog box, which displays a list of all runs that include valid standard curves that can be imported into the displayed run.

• Compare Run — opens the Select a Run dialog box, which displays a list of all runs in the database that can be compared. This feature allows you to monitor two runs simultaneously.

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Defining a New Graph

Figure 3-15: Procedure to define a new graph.

Once a new graph has been defined and saved, associate it with the current run as follows (see “Displaying Graphs” on page 90 for more details):

• Click the View Results icon.

• Click the Select Graphs button.

• Highlight the new graph name.

• Click the right arrow and click OK when the selection is complete.

1. Click the Define Graphs icon.

2. Click the New Graph button.

3. Enter a unique graph name and click OK.

4. Check the Automatically added to new Runs box to add the graph to all new runs. Leave unchecked if the graph should not be added to all runs.

5. Select Optics, Temperature, Standard Curve or Melt from the Graph Type drop-down menu.

6. For optics graphs, check up to four Channels to display on the graph.

7. Check the graph attributes to Show on graph.

8. Select the graph axes.

9. Click the Save Graph button.

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6

7

8

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Setting up the Analysis SettingsClick Analysis Settings in the Views list.

Figure 3-16: The Analysis Settings table.

Note: Always click the Update Analysis button after any changes have been made to the Analysis Settings to implement the changes (see “Selecting Analysis Settings” on page 78 for more details).

1. Usage — select Unused, Assay (default), Internal Control or QIC.

2. Bkgnd Sub — select OFF to view raw data, select ON for Ct analysis.

3. Bkgnd Min and Max Cycle — sets the background range for calculat-ing average background.

4. Curve Analysis — select Primary Curve or 2nd Deriv.

5. Threshold Setting — select Man-ual and enter a value in the Manual Thresh Fluor Units column or

select Run-based Auto or Site-based Auto and enter a value in the Auto Thresh #SD’s column and specify the background range in the Auto Min and Max Cycle columns.

6. Valid Min and Max Cycle — enter a valid cycle threshold range.

7. Boxcar Avg Cycle — enter a value between two and five to boxcar average the data.

8. Target — enter name of the target DNA or RNA, optional.

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Viewing the Results Table and Setting Up a Standard Curve

Figure 3-17: The Results Table.

Notes: For a quantitative assay, a minimum of 2 standards with valid Ct and concentrations not equal to zero must be used to generate a standard curve.

For quantitative analysis, values for unknown samples will be calculated from 1/2 log below the lowest standard to 1/2 log above the highest standard. Values outside of this range are designated as ND.

1. Click Results Table in the Views list.

2. Enter a Sample ID, optional.

3. Sample Type — select UNKN (default), STD (standard), or UNUSED.

4. Enter Notes, optional.

5. Status — displays OK, Warning or Error for each site.

6. “DYE” Std/Res — enter standard concentration if Sample Type is STD. Displays POS/NEG result if all

Sample Types are UNKN or quan-titative result if at least 2 valid STD Ct values are reported.

7. “DYE” Ct — displays a Ct value if the primary curve or 2nd derivative peak is above the threshold.

8. Melt Peak 1-5 — displays up to five melt peaks. The number of melt peaks displayed depends on the number designated in the default melt settings. See “Setup Menu” on page 57 for more details.

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Printing a Report

Figure 3-18: A Run Report.

1. Click the Report button at the bot-tom of the View Results screen.

2. Click the First, Previous, Next or Last buttons to scroll through the report pages.

3. Click the Print button to print the report.

4. Click the Close button to return to the View Results screen.

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Exporting Run Data

Figure 3-19: The Export Data dialog box.

Notes: Do not delete the “.csv” after the file name.

A shortcut to the SmartCycler Export folder will be placed on the desktop during installation.

Create a subfolder for each user in the Export folder so that each user can export runs to a personal folder.

1. Click the Export button at the bottom of the View Results screen.

2. Check the type of data to export.

3. Click the Export button in the Export Data dialog box.

4. Select a folder from the Look In drop-down menu. The software will default to the Export folder that is located in the SmartCy-cler folder.

5. Enter a file name or accept the default file name.

6. Click Save.

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Chapter 4: The SmartCycler Software

4.1 Introduction to the SmartCycler Software . . . . . . . . . . . . . . . . . . 53• SmartCycler Software Screens . . . . . . . . . . . . . . . . . . . . . . . 54

4.2 SmartCycler Software Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55• User Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55• Logs Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56• Setup Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57• Tools Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64• Help Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

4.3 The Create Run Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70• Creating a Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71• Viewing Run Setup Reports. . . . . . . . . . . . . . . . . . . . . . . . . . 74• Cancelling Run Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74• Selecting Graphs to Associate with the Run . . . . . . . . . . . 74• Copying Run Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

4.4 The View Results Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76• Selecting Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . 78• Setting a Threshold Manually . . . . . . . . . . . . . . . . . . . . . . . . . 81• Setting a Threshold Automatically . . . . . . . . . . . . . . . . . . . 82• Setting a Valid Cycle Threshold Range . . . . . . . . . . . . . . . . 84• Boxcar Averaging Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

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• Viewing the Results Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85• Viewing Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90• Displaying Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90• Selecting Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95• Comparing Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96• Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96• Viewing and Printing Reports . . . . . . . . . . . . . . . . . . . . . . . . 99• Importing a Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . 100

4.5 The Define Protocols Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104• Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104• Defining a Thermal Cycling Protocol. . . . . . . . . . . . . . . . . . 106• Defining a Protocol with a Melt Curve Stage. . . . . . . . . . . 109• Using the Advance to Next Stage Feature . . . . . . . . . . . . . 110• Editing Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

4.6 The Define Graphs Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114• Creating a New Temperature Graph . . . . . . . . . . . . . . . . . . 115• Creating a New Optics Graph . . . . . . . . . . . . . . . . . . . . . . . . 116• Creating a New Standard Curve Graph . . . . . . . . . . . . . . . . 119• Creating a New Melt Graph . . . . . . . . . . . . . . . . . . . . . . . . . . 120• Duplicating a Graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121• Editing Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122• Deleting Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

4.7 The Stop Run Button. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

4.8 The Check Status screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

4.9 The Maintenance Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 • Error Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

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Introduction to the SmartCycler Software

4.1 Introduction to the SmartCycler SoftwareThe SmartCycler Software has five menus and seven major screens, represented by icons across the top of the interface.

Figure 4-1: The SmartCycler Software user interface.

Click on the icons across the top of the interface to change screens; the icon for the selected screen is highlighted. If a screen is unavailable because a processing block is not attached or turned on or a run has not been started, the icon is grayed and cannot be selected. For example, in Figure 4-1, the Check Status and Stop Run icons are grayed because no runs have been started since the software was opened and the Create Run icon is grayed because no instrument is connected.

Some of the SmartCycler Software screens are subdivided into sets of panels by sliding dividers (see Figure 4-1). To resize a panel, drag the dividers that adjoin it.

The functions and features of each screen are summarized below. Detailed explanations of each menu and screen are found later in this chapter.

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SmartCycler Software ScreensCreate Run — select to define a new run. The Run Name, Dye Set, Sites, and Protocol(s) must be specified before the run is started. The Start Run button is located in the bottom left corner of this screen. The user can also select how to analyze the data (Analysis Settings), setup standard curves (Results Table), select graphs, and add sample information.

View Results — select to view results in real time. The user can select how to analyze the data (Analysis Settings), setup standard curves (Results Table), select graphs, and add sample information. Other functions such as exporting data, printing reports, viewing other runs, comparing runs and importing standard curves are also performed in this screen.

Define Protocols — select to create, modify, rename, duplicate and delete protocols.

Define Graphs — select to define new graphs and edit existing graphs. A graph definition includes the graph name, Graph Type (e.g. optics, temperature), and optics graph choices such as Channel(s), which curves to show (Primary Curve and/or 2nd Derivative) and what axis type (fluorescence or log-fluorescence) to use for the optics graphs.

Stop Run — select to terminate entire runs or individual sites before the run has finished.

Check Status — select to monitor the status of experiments in real time. This screen provides tables of information for each run that has been started since the software was opened and each site, including all protocols in use and an estimate of time remaining. The Site ID, cycle Status, Protocol, and I-CORE status are provided for each site.

Maintenance — select to view information about the I-CORE modules, the SmartCycler processing block serial number, the current user, the software version and any error or warning messages.

The SmartCycler Software stores all saved protocols, runs, and graphs in a database. This allows the user to view run data at any time for analysis, exporting, or printing. It also enables the user to re-use any saved run setup, protocol or graph, eliminating the need to re-create the settings. Saved runs

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SmartCycler Software Menus

can be archived to an alternate location such as a network drive, hard-drive or other large storage medium for backup purposes. See “Tools Menu” on page 64 for detailed information on database functions. The order in which graphs and protocols are listed can be customized to allow easier access to frequently-used items. See section 4.3, “The Create Run Screen” on page 70 for infor-mation on creating runs; section 4.5, “The Define Protocols Screen” on page 104 for information on creating, modifying, deleting and re-ordering protocols; and section 4.6 “The Define Graphs Screen” on page 114 for information on creating, modifying, deleting and re-ordering graphs.

4.2 SmartCycler Software Menus

Figure 4-2: The main SmartCycler Software menu.

User Menu

Figure 4-3: The User menu.

• Login — if user administration has been activated (by defining users), select to log in if the software is already open. When the software is opened, all users must log in if any users have been defined.

• Change Password — select to change your password.

• Logout — select to log out without closing the software. It is good prac-tice to log out if the SmartCycler System is unattended. Logging out will not affect the run in progress.

• Exit — select to close the SmartCycler Software.

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Logs Menu

Figure 4-4: The Logs menu.

• Run Log — select to search and create a log of runs based on the run date, user name, protocol name or reagent lot number.

Figure 4-5: The Select Criteria for Run Log dialog box.

• Specimen Report — select to search and create a report based on the sample ID.

Date — select all dates or click Select and enter a range of dates.

Users — select all users or click Select and check a specific user name(s).

Protocols — select all protocols or click Select and check a specific protocol(s).

Lot Number — enter a lot number. To search for a lot number use “_” as a single character wildcard, or “%” for any number of characters.

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Figure 4-6: The Select Criteria for Specimen Report dialog box.

Note: If more than 150 sample IDs match the selection criteria only the first 150 will be shown in the report.

The Run Log and Specimen Report include the Run Name, User Name, Run Status (Done or Stopped), Started At (date and time), Finished At (date and time), software Version, Protocol, Lot Number. The Specimen report also includes the Protocol, Site ID, Sample Type, “DYE” Results, and Notes.

Setup Menu

Figure 4-7: The Setup menu.

Date — select all dates or click Select and enter a range of dates.

Specimen Sample ID — enter a sample ID. To search for a sample ID use “_” as a single character wildcard, or “%” for any number of characters.

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User Administration

User administration functions allow you to set up user names, passwords and user access rights to the SmartCycler Software features. User administration can be used to limit access to specific software features or to track users. See Appendix A “User Administration” on page 197.

Figure 4-8: The User Administration dialog box.

System Defaults

• General — select to set the location name of the SmartCycler System, select paper size, and date format (order of month, day, and year) for logs and reports.

Note: General system defaults are normally set for an entire company or lab. They should not be changed for each user. If user administration is activated, only users with System Administrator rights have access to the General defaults.

• Analysis Settings — select to set the default analysis settings for the entire system. The new default analysis settings will be applied to all new runs for all users. It is not possible to set unique default analysis settings for each user.

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Figure 4-9: Setting new default Analysis Settings.

Note: Always click the Cancel Run Setup button at the bottom of the Create Run screen after changing default Analysis Settings.

• Automatic Backup — select to set a backup reminder or enable the auto-matic backup feature. See “Working With Automatic Backup” on page 60 for more details.

• Export Settings — select to set the default export settings for the entire system and enable automatic export. All of the defaults entered in this window will be applied to all runs. See “Export Settings” on page 61 for more details.

• Melt Settings — select to set default melt settings for the entire system. See “Melt Settings” on page 63 for more details.

• Access Options — select to specify whether users can access only their own runs, protocols and graphs or access all runs, protocols and graphs.

Figure 4-10: The Access Options dialog box.

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Note: User administration must be activated to use this feature. Make sure that all users do not have system administration rights because the system administrator can access all runs, protocols and graphs even if this feature is activated.

Working With Automatic Backup

Figure 4-11: The Automatic Backup dialog box.

To Set a Backup Reminder

1. Select Give backup reminder every __ days for the software to automati-cally display a reminder to perform a backup at a defined time interval.

2. Enter a value between 1 and 365 days.

To Enable Automatic Backup

1. Select Perform automatic backup to the specified path or file name below to perform an automatic backup of the database at a defined time interval.

2. Enter a value between 1 and 365 in the Occurs every __ days field.

3. Accept the default folder (C:> SmartCycler> Backup) or click the Browse button and select a different default folder into which the backup file will be saved.

4. Enter a file name to write to the same file every time. This will overwrite the previous backup file each time a new backup is created. If this field is left blank a new file will be created each time a backup is performed.

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Notes: If the same file name is used each time a backup is created the previous backup file will be overwritten. To avoid overwriting the previous backup file, rename the most current backup file before the scheduled backup occurs.

If a new file is created every time a backup is performed and saved on the local drive, it will be necessary to periodically delete old files.

Export Settings

Figure 4-12: Setting default Export Settings.

• Data Export Defaults — check the type of data to export.

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Notes: If the Automatic export option has been selected only the defaults selected in this window will be exported.

If the data is exported manually it is possible to change the type of data to export in the Export Data dialog box (View Results screen). However, changes to the Optics Data defaults and Melt Data defaults must be made in the Export Settings dialog box (Figure 4-12).

• Optics Data Defaults — check to export primary curve or 2nd derivative data or select both.

• Melt Data Defaults — check to export melt curve or 1st derivative data, or select both.

• Export Path and Filename Defaults — click the Browse button to select a different default folder to save all export files. The default folder is C:> SmartCycler> Export. If a file name is entered the software will automat-ically write to the same file name every time and the previous file will be overwritten every time the data is exported. If this field is left blank a unique default file name will be used.

• Automatic Export Option — select to automatically export the run data after the run is complete.

Note: If a new file is created every time data is exported and the files are saved on the local drive, it will be necessary to periodically delete old files.

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Melt Settings

Melt Settings specify default melt settings for the entire system.

Figure 4-13: The Melt Settings dialog box.

• Smooth the melt curve by averaging between 1 and 15 points. Select the number of points to average from the drop-down menu.

• Show a maximum of one to five melt peaks. This value sets the number of melt temperatures that will be displayed on the graph and the number of melt temperature columns that will appear in the Results Table.

Note: If you choose to show five melt peaks and only two peaks meet the acceptance criteria, then only two melt temperatures will appear on the graph and in the Results Table.

• Set the minimum acceptable height of the melt peak. The valid range is 1–100 units. The default is 10 units. Set the threshold above noise peaks but low enough to detect weak positives. If the height cutoff is set too low, noise peaks will be included in the results.

Note: Increase the acceptable peak height value if noise peaks are crossing the default threshold of 10 units. Decrease this value if true peaks are below the default threshold of 10 units.

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• Set the valid temperature range for the melt peaks. For example, if the valid range is between 65 degrees and 100 degrees, melt peaks outside of this range will not be included in the results.

Tools Menu

Figure 4-14: The Tools menu.

Data Management

Archive Run(s) — select to archive one or more runs to a file. Archived runs can be retrieved back into the database at any time. Archived runs are approxi-mately 2 MB per run. It is recommended to store archive files on a network drive or other large storage medium.

Note: Runs cannot be archived directly to a network. To save archived files on a network, first archive the runs to the local drive (C drive) then copy the file(s) to the network drive.

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Figure 4-15: Archiving runs.

Retrieve Run(s) — select to retrieve one or more runs from an archive run file.

Note: Runs cannot be retrieved directly from a network or CD. Copy the archive file to the local drive (C drive) then proceed with retrieve process.

1. Select Tools > Data Management > Archive Runs (s).

2. Click Proceed.

3. Select up to 1000 runs to archive by highlighting the run name(s). Use the [Shift] key to select con-tiguous runs or the [Ctrl] key to select non-contiguous runs. To sort by Name, Status, User or Date, click on the corresponding column header.

4. When all desired runs have been selected, click OK.

5. Select Proceed to continue or Cancel to discontinue the process.

6. Select a folder and enter a file name or accept the default. Click Save.

7. The Archive dialog box will appear when the runs have been successfully archived.

Important:Archiving does not remove runs from the database.

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Figure 4-16: Retrieving an archived run file.

Complete Backup — select to create a backup copy of the entire database. The backup copy can be saved to the local hard drive, a network, or a large storage medium. The backup file must be saved to a local drive first then copied to a

1. Select Tools > Data Manage-ment > Retrieve Runs (s).

2. Click Proceed.

3. Select the archive file name to retrieve and click Open.

4. Select the run(s) to retrieve by highlighting the Run Name and clicking OK. Runs that already exist in the cur-rent database will be red and in italics. Use the [Shift] key to select contiguous runs or the [Ctrl] key to select non-contiguous runs. To sort by Name, Status, User or Date, click on the corresponding column header.

5. If a red italic Name is selected, the archived run will overwrite the run in the data-base. Click Proceed to con-tinue with the process. The software will close at the end of the retrieve process.

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network drive or other large storage medium. In the event of a major computer failure, the database can be restored from the backup copy.

To manually back up a database

1. Select Tools > Data Management > Complete Backup.

2. Click Proceed in the Database Backup dialog to continue the process.

Figure 4-17: The Database Backup dialog box.

3. Select a folder from the Look in drop-down menu.

4. Enter a file name or accept the default file name and click Save.

Figure 4-18: Saving the backup file.

5. The software will confirm that the database has been backed up.

Figure 4-19: The Database Backup dialog box, confirming the backup has been completed.

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Complete Restore — select to restore an entire database from a backup copy. If a database is restored, the current database will be replaced and all runs, protocols and graphs will be lost. Do not restore a database unless the current database is corrupted or needs to be replaced.

Important:Back up the current database prior to overwriting it!

To completely restore a database

1. Select Tools > Data Management > Complete Restore.

2. Click Proceed in the Database Restore dialog to continue the process.

Figure 4-20: The Database Restore dialog box.

3. Select a folder from the Look in drop-down menu.

4. Select the backup file name and click Open.

Figure 4-21: Selecting a file to restore.

5. The software will confirm that the database has been restored.

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Figure 4-22: The Database Restore dialog box, confirming the restoration has been completed.

Compact Database — select to compact the database by removing unused space from the database.

Optical Calibration — select to calibrate the I-CORE modules with dyes other than FAM, Alexa Fluor 532, Cy3, TET, Texas Red, Cy5 or Alexa Fluor 647. See Appendix B “Optical Calibration” on page 203 for details.

Save Screen to File — select to save the current screen as a JPEG file.

1. Select a folder from the Look in drop-down menu (the default folder is the Export folder located in the SmartCycler folder).

2. Enter a file name for the JPEG file in the File name field.

Figure 4-23: Saving a screen as a JPEG file.

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Help Menu

Figure 4-24: The Help menu.

Help — select to open the online help guide.

About — select to display information about the software version and copyright warnings.

4.3 The Create Run Screen

Figure 4-25: The Create Run screen.

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The Create Run Screen

Note: All information entered in the Create Run screen is automatically saved until the run setup is cancelled or the SmartCycler Software is closed. Once a run has started, the run name, user, dye set, sites, protocols and start time cannot be changed.

Creating a Run

Figure 4-26: The left panel of the Create Run screen.

1. Click the Create Run icon.

2. Enter a unique Run Name. Run names must not contain the following charac-ters: ^ | < > : * ? \ /.

3. Enter Notes about the new run. Notes can be edited before, during or after a run has completed.

4. Click the Dye Set arrow and select a dye set from the drop-down menu. A dye set specifies the dye specific calibration data. See Figure 4-27 for the Dye Set drop-down menu.

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Figure 4-27: The Dye Set drop-down menu.

If the Dye Set is changed after protocols and sites have been selected, the run setup information will be cleared and the following message will appear (Figure 4-28).

Figure 4-28: The Change Dye Set dialog box.

5. Click the Add/Remove Sites button to open the Select Protocols and Sites dialog box and select protocols and sites for the new run.

Figure 4-29: The Add/Remove Sites button.

• FCTC25 — FAM, Cy3, Texas Red, Cy5, 25 μL reaction tube.

• FATA25 — FAM, Alexa Fluor 532, Texas Red, Alexa Fluor 647, 25 μL reaction tube.

• Intcltr_dye — intercalating dye, 25 μL reaction tube. Note: Ethidium bromide cannot be used as an intercalating dye in the Smart Cycler system.

• FTTC25 — FAM, TET, Texas Red, Cy5, 25 μL reaction tube

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Figure 4-30: The Select Protocols and Sites dialog box.

Note: To assign a protocol to multiple sites use the [Shift] key to select contiguous sites and the [Ctrl] key to select non-contiguous sites or click the Select All Sites button to add all sites.

10. To assign multiple protocols, repeat steps 6–8 for each protocol until all protocols and sites have been selected.

11. Enter sample IDs, identify sites as standards by clicking in the sample Type column and selecting STD from the drop-down menu and entering standard concentrations in the “DYE” Std Conc field.

Figure 4-31: The sites table in the Create Run screen.

12. Make changes to the analysis settings before the run is started. See “Select-ing Analysis Settings” on page 78 for more detailed information.

6. Select a protocol name from the Protocols list.

7. Select site(s) from the available Sites list.

8. Click the right-point-ing arrow to transfer the assigned sites to the Selections field.

9. Click OK when the selection is complete and correct.

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Figure 4-32: Making changes to the Analysis Settings in the Create Run screen.

Important:The analysis settings must be set up before the run is started if the Advance to Next Stage feature is enabled.

13. Click Start Run to start the new run. This button will be grayed and inactive until the site(s) and protocol(s) have been assigned.

Note: All Analysis Settings, Results Table entries and graph selections can be set up before, during or after the run has completed unless using Advance to Next Stage. Only a unique run name, dye set, protocol and site selections are required before a run has started. Please see section 4.4, “The View Results Screen” on page 76 for more details.

Viewing Run Setup Reports1. Click the Report Run Setup button before the run has started to view or

print a report detailing setup parameters for the new run.

Cancelling Run Setup1. Click the Cancel Run Setup button to reset the run setup information.

Selecting Graphs to Associate with the Run1. Click the Select Graphs button to select graphs from the database to asso-

ciate with the run.

2. If the desired graph is not listed follow the procedure in section 4.6 “The Define Graphs Screen” on page 114 to create a new graph.

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Copying Run Setup1. Click the Copy Run Setup button to copy the complete setup of a previous

run.

2. Select a run from the Select a Run dialog box.

Figure 4-33: The Select a Run dialog box.

3. All of the run information will be copied into the new run including the run name, dye set, sites and sample IDs.

4. It is not possible to start a run with having the same name as an existing run; it is necessary to re-name the new run before it can be started.

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4.4 The View Results Screen


The far left section of the View Results screen displays information entered/selected in the Create Run screen. Only the information in the Notes and Lot Number fields can be edited.

The buttons across the bottom of the screen have the following functions:

• Save Run — click to save any changes made to the Notes, Lot Number, Analysis Settings, or Results Table before switching screens, printing reports, exporting data, or closing the SmartCycler Software.

If you attempt to switch screens or exit the software before changes have been saved, the Save Changes? dialog box will appear.

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The View Results Screen

Figure 4-35: The Save Changes? dialog box.

Note: The SmartCycler Software does not give a prompt to save changes before printing or exporting. Therefore, it is possible to make changes to the Results Table or Analysis Settings, and immediately print or export the data, then close the run without saving the changes. This would cause the data saved in the SmartCycler database to not match the printed or exported data.

• Export — click to export optical, melt, or temperature data, or Analysis Settings and Results Table. See “Exporting Data” on page 96.

• Report — click to display and print a run report. Run reports cannot be edited.

• Select Graphs — click to add or delete graphs from a run. See “Displaying Graphs” on page 90.

• View Another Run — click to display the run names in the database and select a run to view.

Note: Click on a column header in the Select a Run dialog box to sort the runs by the Run Name, Status, User, or Date.

• Delete Run(s) — click to delete one or more runs from the database. Mul-tiple runs can be deleted at one time.

Note: Click on a column header in the Delete Run(s) dialog box to sort the column by the Run Name, Status, User, or Date.

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• Update Analysis — click to implement changes made to the Analysis Settings and Results Table.

• Import Std Curve — click to display a list of runs that include standard curves. For detailed information about quantitating unknowns in one run with a standard curve from another run. See “Importing a Standard Curve” on page 100.

• Compare Run — click to compare two runs. See “Comparing Runs” on page 96.

When an option is selected from the Views lists, the corresponding view is displayed to the right of the list.

Figure 4-36: Displaying views.

Selecting Analysis Settings

Figure 4-37: The Analysis Settings table.

Select Analysis Settings from Views to edit or display how the data for each dye channel will be analyzed. Analysis settings include one row for each of the four dye channels defined in the dye set.

Click the Update Analysis button to implement changes made to the analysis settings.

Click the Save Run button to save changes made to the analysis settings.

Selected view

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Note: Always click the Save Run button to save changes to the analysis settings before printing or exporting data.

Important: Analysis settings are applied to the raw data, but do not change the raw data. Analysis settings can be changed before, during or after the run is finished.

Figure 4-38: The left side of the Analysis Settings pane.

• Ch # — the channel listed in the dye name column (e.g., Ch 1 = FAM). This column is not editable.

• Dye Name — the dye name as defined by the dye set. This column is not editable.

• Usage — select the channel usage (Unused, Assay, Internal Control, or Quantitative Internal Control (QIC)) from the drop-down menu. One channel per run can be designated as an Internal Control or QIC.

Note: If a channel is designated as Unused, it will not be displayed in the Results Table, exported data, graphs or run reports.

• Bkgnd Sub — click in the table cell to select ON or OFF from the drop-down menu. To view raw signal select OFF. To view background sub-tracted data and to analyze data select ON. This setting will be applied to all channels. See “Background Subtraction” on page 29 for more details.

• Bkgnd Min Cycle — the first cycle used to calculate the background if background subtraction is ON. The default value is 5. Decrease this value if the cycle threshold occurs before cycle 13.

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• Bkgnd Max Cycle — the last possible cycle used to calculate the back-ground if background subtraction is ON. The default value is 40.

• Curve Analysis — select to analyze the primary curve or the 2nd deriva-tive curve. The default setting is Primary Curve. See section 3.3, “Cycle Threshold” on page 35 for more details on selecting the curve analysis.

Figure 4-39: The Curve Analysis drop-down menu.

• Thresh Setting — select Manual, Run-based Auto, or Site-based Auto from the Threshold Setting drop-down menu. Site-based Auto is only available if the Curve Analysis is set to 2nd Deriv. See “Setting a Thresh-old Manually” on page 81 for more details on using this feature.

• Manual Thresh Fluor Units — specifies a threshold setting manually. See “Setting a Threshold Manually” on page 81 for more details on using this feature.

• Auto Thresh # SD’s — when using automatic threshold detection, the standard deviation of the fluorescence signal within the range of cycles defined by the Auto Min Cycle and Auto Max Cycle value is calculated for each site. The largest standard deviation of all the sites is multiplied by the number in the Auto Thresh # SD’s column to define the threshold. See “Setting a Threshold Automatically” on page 82 for more information.

• Auto Min Cycle and Auto Max Cycle — when using automatic threshold detection the standard deviation of the fluorescence signal within the range of cycles defined by the Auto Min Cycle value (default = 5) and the Auto Max Cycle value (default = 10) is calculated for each site to measure the background noise. See “Setting a Threshold Automatically” on page 82 for more information.

• Valid Min Cycle and Valid Max Cycle — define, respectively, the earliest and latest earliest cycle numbers within which the threshold crossing is considered valid. See“Setting a Valid Cycle Threshold Range” on page 84 for more information.

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• Boxcar Avg Cycles — specifies the number of cycles to be used in boxcar averaging of the data. See “Boxcar Averaging Data” on page 84 for more information.

• Target — (optional) enter the DNA/RNA target name or the assay name. If a target name is entered in this field, the Results Table will display the target name instead of the dye name in the column header.

Setting a Threshold ManuallyThe manual threshold setting is specified by entering a fluorescent unit value in the Manual Thresh Fluor Units cell or by clicking the square box on the end of the threshold line on the graph and dragging the line up or down. The manual threshold should be set such that it intersects the growth curve at the first significant increase in fluorescence.

Note: The default setting of 30 may not be optimal for every data set. Increase or decrease the threshold value according to your data set.

• Select Manual (default) to set the threshold at a specific fluorescent unit value. Enter a fluorescence unit value or accept the default of 30 fluores-cent units in the Manual Thresh Fluor Units column (Figure 4-40).

• Alternatively, click the square box on the end of the graph’s threshold line and drag the line up or down until it intersects the growth curve at the first significant increase in fluorescence (Figure 4-41).

Figure 4-40: Manually setting the threshold via the Thresh Setting drop-down menu.

Enter desired threshold

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Figure 4-41: Manually setting the threshold by dragging the horizontal threshold on graph.

Setting a Threshold Automatically

Using a Run-based Auto Threshold Setting

The SmartCycler Software calculates the standard deviation of the fluores-cence signal within the range of cycles defined by the Auto Min Cycle value (default = 5) and the Auto Max Cycle value (default = 10) for each site to measure the background noise. The largest standard deviation of all the sites is multiplied by the number in the Auto Thresh # SD’s column to define the threshold.

Figure 4-42: Run-based automatic threshold setting.

1. Select Run-based Auto from the Thresh Setting drop-down menu.

2. Enter the number of standard deviations above background to calculate threshold in the Auto Thresh # SD’s column. The default value is 1.

3. Enter minimum cycle used to calculate background for the threshold deter-mination in the Auto Min Cycle column. The default value is 5.

4. Enter the maximum cycle used to calculate background for threshold deter-mination in the Auto Max Cycle column. The default value is 10.

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Using a Site-based Auto Threshold Setting

The SmartCycler Software calculates the standard deviation of the fluores-cence signal within the range of cycles defined by the Auto Min Cycle value (default = 5) and the Auto Max Cycle value (default = 10) for each site to measure the background noise. The standard deviation for each site is multi-plied by the value in the Auto Thresh #SD’s column to define a threshold for that site. The Site-based Auto threshold setting can only be used if the Curve Analysis is set to 2nd Deriv. Each site will have a different threshold.

Figure 4-43: Site-based Auto defines a threshold for each site.

1. Select Site-based Auto from the Thresh Setting drop-down menu.

2. Enter the number of standard deviations above background to calculate threshold in the Auto Thresh # SD’s column. The default value is 1.

Figure 4-44: Site-based auto threshold setting.

3. Enter minimum cycle used to calculate background for the threshold deter-mination in the Auto Min Cycle column. The default value is 5.

4. Enter the maximum cycle used to calculate background for threshold deter-mination in the Auto Max Cycle column. The default value is 10.

A threshold is defined for each site

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Note: See section 3.3, “Cycle Threshold” on page 35 for more details on setting a threshold.

Setting a Valid Cycle Threshold RangeA valid cycle threshold range is most often used for setting a valid range for an internal control but this feature can be used for channels designated as Assay or QIC.

1. Enter the earliest cycle number that the threshold crossing is considered valid in the Valid Min Cycle column. This value sets the minimum valid cycle threshold. To change the default of 3, highlight the cell and enter a new value.

Figure 4-45: Setting a Valid Min Cycle and Valid Max Cycle range.

2. Enter the latest cycle that the threshold crossing will be considered valid in the Valid Max Cycle column. This value sets the maximum valid cycle thresh-old. To change the default of 60, highlight the cell and enter a new value.

Boxcar Averaging DataBoxcar averaging can be used to reduce noise in the data and smooth the growth curves. Boxcar averaging can change the cycle threshold value slightly because the data points are being averaged. If boxcar averaging is used in one run it should be used in all runs that will be compared.

1. Enter a value between 2 and 5 in the Boxcar Avg Cycles column (Figure 4-46). The default value is “0” (OFF).

Note: Use the lowest value that produces acceptable noise levels.

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Figure 4-46: Boxcar averaging data.

Viewing the Results TableClick Results Table in the Views list to display run results, enter Sample ID’s and set up a standard curve.

Figure 4-47: The Results Table.

• Site ID — displays ID of processing block (A, B, C, or D) and site number (e.g., A5). Values in this column cannot be modified.

• Protocol — displays the name of the protocol assigned to each site. This column cannot be modified.

• Sample ID — (optional) enter an ID or description of the sample.

• Sample Type — specifies the sample type. See “Selecting Sample Types” on page 86 for more information. This column is used in setting up a standard curve; see “Setting Up a Standard Curve” on page 87 for more information.

• Notes — can be used to enter notes about each sample.

• Status — displays the current status of the site. OK will be displayed if no errors or warnings have occurred. Error or Warning will be displayed if the site reported an error or warning. If the site reported an error or warning, go to the Maintenance screen to view the error or warning code and description. See “Warning Messages and Error Codes” on page 193.

• “Dye” Std/Res (standard concentration/results) — when the Sample Type is UNKN for all sites this column displays POS if a threshold crossing was detected in the valid range or NEG if threshold crossing was not detected in the valid range. If there are at least two valid standards with non-zero

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concentrations in the run, this column will display the calculated sample concentration for any UNKN samples included in the run. When the Sam-ple Type is set to STD, the concentration of each standard is entered in the “DYE” Std/Res column. See “Setting Up a Standard Curve” on page 87 for more information. If the Sample Type is UNUSED the column will dis-play No Result. See “Selecting Sample Types” on page 86 for more infor-mation.

• “Dye” Ct — the “Dye” Ct column will display the Ct (cycle threshold) value for channels designated as Assay or Internal Control in the analysis settings. See “Viewing Cycle Threshold Results” on page 89 for more information.

• Melt Peak — displays melt peak data for the sample. See “Viewing Melt Peak Results” on page 89 for more information.

Click the Update Analysis button at the bottom of the View Results screen to implement changes made to the Results Table.

Click the Save Run button at the bottom of the View Results screen to save changes made to the Results Table before printing or exporting data.

All changes made to the Results Table only affect how the data is analyzed and displayed; the raw data is not changed.

The Results Table displays columns for all channels except channels designated as Unused in the analysis settings (see “Selecting Analysis Settings” on page 78).

Selecting Sample Types

Use the Sample Type column to define each site as an unknown (UNKN), standard (STD) or unused (UNUSED). Click in the Sample Type column cell for each site, then select UNKN, STD, or UNUSED from the drop-down menu.

Figure 4-48: The Sample Type drop-down menu.

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If the Sample Type is UNKN for all sites the “DYE” Std/Res (standard concen-tration/results) column displays POS if a threshold crossing was detected in the valid range or NEG if threshold crossing was not detected in the valid range.

Figure 4-49: “Dye” Std/Res results for samples when all sample types are UNKN.

When a Sample Type is set to STD, enter the concentration of each standard in the “DYE” Std/Res column (acceptable range=0.001–1.0E9). If there are at least two valid standards with non-zero concentrations in the run, the “DYE” Std/Res column will display the calculated sample concentration for any UNKN samples included in the run (see “Setting Up a Standard Curve”, below).

If the Sample Type is UNUSED the site will not be included in the graphs, calcu-lation of the standard curve or the standard deviation used for setting the automatic threshold. The “DYE” Std/Res column will display No Result.

Setting Up a Standard Curve

1. Select Results Table in the Views list. Set up the Results Table table as fol-lows for each site:

Figure 4-50: Setting up standards in the Results Table.

2. Select STD from the Sample Type drop-down menu for each standard.

3. Enter the concentration (i.e., copy number of DNA or cDNA) of each stan-dard in the “DYE” Std/Res column. The acceptable range is 0.001 to 1.0E9.

4. Click the Update Analysis button.

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5. Select Standard in the Views list. The Standard graph plots standards (STD) as blue diamonds and unknowns (UNKN) as red rectangles.

Figure 4-51: Viewing a standard curve.

Note: y = ax+ b, r-squared = c (where a, b, and c are all numbers). a defines the slope, b is the Y intercept and r-squared is the correlation coeffi-cient, which defines how well the standards fit to a straight line.

6. It is possible to transpose the X and Y axes by right-clicking on the graph and selecting Transpose X and Y Axes from the drop-down menu.

Figure 4-52: Transposing the x and y axes of a standard curve graph.

Unknown Standard

Calculated linear fit Correlation coefficient

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Viewing Cycle Threshold Results

The “DYE” Ct column will display the Ct (cycle threshold) value for channels designated as Assay or Internal Control in the analysis settings.

Figure 4-53: The “DYE” Ct column when all channels are Assay or Internal Control.

If one channel is designated as QIC, the “DYE” Ct column for the QIC channel will display Ct values and the Assay channels will display ratios (see section 5.6, “Overview: Running a Multiplex Assay with a Quantitative Internal Control (QIC)” on page 160).

Figure 4-54: The “DYE” Ct column when one channel is designated as QIC.

Viewing Melt Peak Results

The number of melt peak columns displayed in the Results Table is specified in the Melt Setting defaults. To add or remove melt peak columns, select Setup > System Defaults > Melt Settings. See “System Defaults” on page 58.

Figure 4-55: Viewing melt peak data.

“Dye” or TargetCt column

“Dye” or TargetCt column

Assay QIC

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The melt peak columns display up to five melting temperatures. The Melt Peak 1 column displays the highest peak value.

If noise peaks are reported as melt peaks, increase the melt peak threshold. To change the default value of 10, select Setup > System Defaults > Melt Settings. If weak positive melt peaks are not detected, decrease the melt peak threshold. The minimum value is 1 unit.

Viewing Protocols

Figure 4-56: A protocol displayed in the View Results screen.

Select Protocols in the Views list to display the protocol(s) used in the run (Figure 4-56). Protocols cannot be edited in the View Results screen (see section 4.5,“The Define Protocols Screen” on page 104).

Displaying GraphsAll graphs associated with the selected run are listed under Views. To display a graph, click the graph name. The graph legend displays a color-coded Site ID, Protocol and Sample ID for each sample (see Figure 4-57).

Note: Colors assigned to the sites cannot be edited. If the same graph is selected in both Views lists, the color keys will be the same for both.

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Figure 4-57: A temperature graph, displayed in the View Results screen.

The sliding dividers can be moved to enlarge the graph display for better viewing or printing.

Graph Options

Right-click on a graph to access a menu of display options.

Figure 4-58: The graph options menu.

• Reset zoom — resets the graph to the default scale. To zoom in on a sec-tion of a graph, click on the graph and move the cursor to draw a box around the desired area. Press the R key or right-click the graph and select Reset zoom to return the graph to the default scale.

• Show all sites — select to display all of the sites on the graph.

• Scale graph — to rescale the graph axes, right-click on the graph and define the min and max limits in the Axes Graph Scale dialog box Figure 4-59).

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Figure 4-59: The Axes Graph Scale dialog box for rescaling graph axes.

Note: Graphs will always be reset to the autoformatted scale each time new data is collected, therefore it is recommended to wait until the run is finished before zooming, scrolling or manually scaling graphs.

Always enter valid values in the Min or Max fields of the Manual Axes Scaling dialog box (i.e., the Min value should always be less than the Max value).

If the graph is zoomed and the graph data is exported (Export graph data) all of the graph points will still be exported.

• Overlay graphs — to overlay the graphs in the two view panels, right click one of the graphs and select Overlay Graphs from the graph options menu. The graph on which you have is right-clicked will be Figure 1 in the Overlay Graph Display window.

The scaling will not be re-adjusted, so each graph will be displayed in the overlay window at its current scale. It is recommended to set both graphs to the same scale before overlaying.

The top frame of the Overlay Graph Display window will display both of the graphs at 100% intensity. To adjust the intensity of the graphs use the intensity% scroll bars. If the intensity is decreased the graph will fade.

The graph overlay window cannot be printed. It is possible to take a screen shot by pressing [Alt] + [PrtScn] and pasting the clipboard image into MS Word or Paint.

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Figure 4-60: Using the Overlay Graph Display window to compare graphs.

• Print — offers options to Print graph only or Print graph with Results Table. Alternatively, the [Alt] + [PrtScn] keys can be used to copy and paste a screen shot of the SmartCycler Software window, including the graph name and the color key, into MS Word or Paint.

• Save to file (jpg) — offers options to save the Graph only or Graph with legend as a JPEG file. Selecting either menu option opens the Save dialog (Figure 4-61). Select a folder from the Look In drop-down menu (the default folder is Export), then enter a file name. Click Save.

Overlay graph

Run name

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Figure 4-61: Saving a graph as a JPEG file.

• Export graph data — saves graph data in a comma separated values (csv) format that can be opened in MS Excel. The exported data will contain only the information that is displayed on the graph.

Note:If the graph is zoomed the software will export the data from the entire graph. If the graph is re-scaled (instead of zoom) the exported data will contain only the data that is displayed on the graph.

Selecting Export graph data opens the Save dialog (Figure 4-61). Select a folder from the Look In drop-down menu (the default folder is Export), then enter a file name. Click Save.

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Figure 4-62: Exporting a graph data file.

Selecting GraphsTo add or remove graphs from a run, click the Select Graphs button at the bottom of the View Results screen to open the Select Graphs dialog box.

Figure 4-63: The Select Graphs dialog box.

1. Highlight one or more graph names in the All Graphs column.

2. Click the right point-ing arrow.

3. The highlighted graph names will be listed in the Selected Graphs column.

4. Click OK when selec-tion is complete.

1 3

2

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Comparing RunsClick the Compare Run button at the bottom of the View Results screen, then select the run to be compared from the Select a Run dialog box.

Note: Click on a column header to sort by Name, Status, User or Date.

The current run will be displayed in the top view. The run that has been selected for comparison is displayed in the bottom view. It is not possible to view another run, switch to another screen, or import a standard curve while two runs are being compared. A dialog box will appear if the user attempts to switch screens, view another run or import a standard curve. It is not possible to save compared runs. If the Save Run button is clicked, only the changes to the current run will be saved. Any changes made to the compared run will not be saved. While runs are compared it is possible to change and update analysis settings for each run and to overlay graphs.

Exporting DataAfter a run is complete the data can be exported into a comma delimited text (.csv) file. To export data:

1. Click the Save Run button before exporting data.

Note: The SmartCycler Software does not give a prompt to save changes before printing or exporting. Therefore, it is possible to make changes to the Results table or analysis settings, and immediately print or export the data, then close the run without saving the changes. In such cases, the data saved in the SmartCycler database and displayed when the run is viewed at a later time will not match the printed or exported data.

2. Click the Export button at the bottom of the View Results screen to display the Export Data dialog box.

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3. Select type of data to export:

• Export Optics Data — by default, Primary Curve and 2nd Deriv data are exported for all channels except channels designated as Unused in the analysis settings.

• Export Results Table and Analysis Settings —exports the Results Table and Analysis Settings as they are displayed in the View Results screen.

• Export Melt Data — exports primary and first derivative melt data by default.

• Export Heater Temperature Data — exports temperature data for each site.

Note: If multiple types of data are exported in a single file, the file will display multiple tables of data in one worksheet.

4. Select a folder from the Look in drop-down menu or accept the default Export folder.

5. Enter a name in the File name field or accept the default file name.

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Figure 4-65: Selecting a folder and entering a name for an export file.

6. Click Save.

Data are exported as comma-delimited text (.csv) files that can be opened with MS Excel. By default, the file is exported to the Export folder in C: >SmartCycler >Export (a shortcut to this folder is installed on the desktop).

When Export Optics Data is selected, the primary curve data and/or 2nd deriv-ative data for each channel will be exported except channels designated as Unused in Analysis Settings (unless default is set up differently). The exported primary curve and 2nd derivative data will reflect the boxcar average cycles and background subtraction analysis settings. For example, if Bkgnd Sub is ON, the background subtracted data will be exported whereas if the Bkgnd Sub is OFF the raw data is exported.

Figure 4-66: An example of exported optics data.

Dye name

Site ID

2nd Derivative

Displays target name (if entered in Analysis Settings) or repeats dye name

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Figure 4-67: An example of exported melt data.

Viewing and Printing Reports Click the Report button at the bottom of the View Results screen to open the Print Preview dialog box. Click the Print button to print the report or use the First, Previous, Next and Last buttons to preview each page of the report.

Figure 4-68: The Print Preview dialog box.

Melt data

1st Derivative

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Note: The SmartCycler Software does not give a prompt to save changes before printing or exporting. Therefore, it is possible to make changes to the Results Table or Analysis Settings, and immediately print or export the data, then close the run without saving the changes, in which case the data saved in the SmartCycler database will not match the printed or exported data.

Importing a Standard CurveIt is possible to quantitate unknown samples with a standard curve from another run by importing the standard curve and its analysis settings into a run that contains only unknowns (UNKN).

Before importing a standard curve, note the Analysis Settings (especially Curve Analysis, Thresh Setting and Boxcar Avg) and the “DYE” Ct results for the current run. Click Analysis Settings and Results Table in the Views list to display the settings and results for the current run.

Figure 4-69: The Analysis Settings and Results Table for the current run.

Importing a Standard Curve and Analysis Settings From Another Run

1. Click the Import Std Curve button, highlight a Run Name from the list of runs that contain valid standard curves and click OK. To sort by Name, Sta-tus, User or Date, click on the corresponding column header.

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Notes: The analysis settings from the standard curve are imported with the standard curve and will temporarily override the analysis settings in the current run.

If a run includes standards or multiple protocols, it is not possible to import a standard curve and the Import Standard Curve button will be unavailable.

The analysis settings cannot be changed while a standard curve is imported. Click the Save Combined Run button or click the Remove Std Curve button, make changes, then re-import the standard curve.

Figure 4-70: Imported standard curve results and quantitation of unknowns.

2. Select Analysis Settings to display how the data for the imported standard curve and current run data are analyzed. The analysis settings cannot be changed while a standard curve is imported.

3. Select Results Table to display the imported standard curve results (high-lighted in yellow) and the current run (UNKN) results.

Analysis settings imported from standard curve run

Analysis settings im-ported from standard curve runStandard

curve run name

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Note: Quantitation of UNKNs is displayed in the “DYE” Std/Res column.

4. Click Standard in the Views list to display the graph of the imported stan-dard curve and quantitated unknowns.

Note: The default Standard graph only displays standards for channel 1. Create new graphs to display standards in other channels.

Figure 4-71: The imported standard curve with quantitated unknowns.

5. Click the Remove Std Curve button to return to the current run.

Saving the Combined Run

1. Click the Save Combined Run button.

2. Enter a new name for the combined run.

Standard (blue)

Imported standard curve

Unknown (red)

Unknowns

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Figure 4-72: Entering a new name for a combined run.

3. Click OK.

Exporting Data From the Combined Run

Follow the procedure detailed in “Exporting Data” on page 96.

The export file name will default to the current run name only. It is recom-mended to add the imported standard curve run name to the file name.

Printing a Report of the Combined Run

To preview or print a report with the imported standard curve click the Report button.

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4.5 The Define Protocols ScreenClick the Define Protocols icon to define and edit SmartCycler protocols in the Define Protocols screen.

Figure 4-73: The Define Protocols screen.

Overview• The Protocol Name list displays all saved protocols (the list will display

only default protocols if no user-defined protocols have been saved yet). Click a protocol name to display the programmed stages.

Note: If user administration and restricted access options have been activated, users will only see their own protocols and default protocols.

• A protocol can have a maximum of ten stages (time/temperature steps or melt curve).

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The Define Protocols Screen

• Each stage can have only one optical read. The optical read step must be at least 6 seconds.

• Each protocol can have a maximum repeat of 100 cycles.

• The acceptable temperature range is 40–98 °C for one processing block operated at room temperature (22–25 °C). If multiple processing blocks are in use or room temperature is higher than 25 °C the minimum temper-ature may increase.

• The maximum run time for a protocol is 14,400 seconds (4 hours). If the run time restriction is exceeded, the SmartCycler processing block will complete the entire protocol, but data from time points beyond the limit will not be recorded.

Note: When calculating how long a protocol will take, remember to include the ramp times between temperatures. For example, if a protocol starts at 60 °C, then increases to 95 °C and holds for 30 seconds, then returns to 60 °C for 20 seconds, it may take close to 5 seconds to warm the site from 60 °C to 95 °C, and close to 15 seconds to cool it from 90 °C to 60 °C. Therefore, each cycle of the stage would take 70 seconds: 5 sec ramp + 30 sec melt + 15 sec ramp + 20 sec anneal/extension.

The buttons across the bottom of the screen have the following functions:

• New Protocol — click to define a new protocol (see “Defining a Thermal Cycling Protocol” on page 106 and “Defining a Protocol with a Melt Curve Stage” on page 109 for more information).

• Delete Protocol — click to delete the selected protocol from the Protocol Name list. Only one protocol can be deleted at a time.

Note: If a protocol that has been used in a run is deleted, the original protocol settings will still be associated with the run.

• Duplicate Protocol — click to create a copy of the selected protocol and add it to the Protocol Name list. The default name for the new protocol

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is “Copy of Protocol X”, where “Protocol X” is the name of the original protocol.

• Rename Protocol — click to rename the selected protocol. Enter a new name in the Rename Protocol dialog box.

• Save Protocol — click to save a new protocol or changes made to an existing protocol. If the Save Protocol button is not clicked after a new protocol is created or changes are made to an existing protocol, the soft-ware will give a prompt to save or discard changes before switching to another screen. The command to switch screens can also be cancelled.

• Move To Top — click to move the selected protocol to the top of the Pro-tocol Name list. The protocols are listed in this order in the Create Run screen’s Select Protocols and Sites dialog box (see section 4.3 “The Cre-ate Run Screen” on page 70 for further information).

Defining a Thermal Cycling Protocol1. Click the New Protocol button. The New Protocol dialog box will open.

Figure 4-74: The New Protocol dialog box.

2. Enter a unique name up to 32 characters (avoid using the following charac-ters: ^ | < > : * ? \ /).

3. Click OK to add the new protocol name to the Protocol Name list.

4. For a Hold stage, select the Hold stage type from the drop-down menu in Stage 1. Enter a Hold temperature (acceptable range for the instrument is 40–98 °C) and the number of seconds the site should remain at that tem-perature (acceptable range is 3–3200 seconds). An optical reading cannot be programmed during a Hold stage.

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Figure 4-75: The Stage Type drop-down menu and a completed Hold stage.

5. For a Temperature Cycle stage, select the desired Temperature Cycle stage type from the drop-down menu in Stage 2.

Figure 4-76: Selecting a Temperature Cycle stage.

6. Enter the number of times to repeat the temperature cycle (integers between 1–100).

7. Accept the default ramp rate (NA) for each step or enter a ramp rate between 0.1–5 in the Deg/Sec column. If the default is accepted the Smart-Cycler will ramp as quickly as possible. Enter a ramp rate only if you want the SmartCycler to ramp more slowly.

8. Enter a temperature (acceptable range is 40–98 °C) for each step of the temperature cycle in the Temp column.

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Note: Do not enter temperatures near or below the processing block internal temperature, or above 98 °C. This will prevent the protocol from progressing, because the I-CORE modules are unable to achieve these temperatures. The processing block internal temperature is displayed in the Maintenance screen (see Figure 4-105).

9. Enter the time (3–3200 seconds) to hold each temperature in the Secs column.

10. Turn the optics On in the annealing or extension step by clicking in the Optics column cell and selecting On.

Notes: The optical read is made at the end of the step for all four channels.

Only one optical read per stage is allowed and the optical read step must be at least 6 seconds.

Figure 4-77: A completed three-stage Temperature Cycle protocol stage.

11. Click the Save Protocol button to save changes.

Note: A warning will appear if a protocol is saved without an optical read. The warning dialog will give the option of continuing the save, or cancelling it to return to the Define Protocols screen.

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Defining a Protocol with a Melt Curve StageIntercalating dyes intercalate all double-stranded DNA including primer-dimers so it is necessary to program a melt curve to immediately follow amplification to discriminate between specific and non-specific products. Melt curves provide information about the purity of the amplification product and the melting temperature of the amplified product(s). Melt curves are generated by slowly ramping up the temperature to 95 °C, and as the DNA melts or becomes single-stranded the intercalating dye is released and the fluorescence signal decreases. The melt curve function can also be used to melt molecular beacons probes. A melt can be programmed from low to high temperature (start 60 °C–end 95 °C) or high to low temperature (start 95 °C–end 60 °C).

Note: Ethidium bromide cannot be used as an intercalating dye with the SmartCycler system.

Defining a Melt Curve Stage after a Temperature Cycle Stage

1. Select Melt Curve from the drop-down menu of stage types.

Figure 4-78: Selecting a Melt Curve stage.

2. Enter the ramp Start temperature (acceptable range is 40–98 °C).

3. Enter the ramp End temperature (acceptable range is 40–98 °C).

4. Select the Optics channel (Ch1 for the intercalating dye).

5. Enter a ramp rate (Deg/Sec) between 0.1 and 1.0 degrees/second.

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Figure 4-79: A Melt Curve protocol stage.

Using the Advance to Next Stage FeatureIf this feature is enabled each site will automatically advance to the next stage of the protocol after a threshold crossing has been detected. The user defines which channel to monitor and how many cycles to wait after a threshold crossing before advancing to the next stage. Only one channel can be monitored for a threshold crossing. For example, if a PCR assay uses an interca-lating dye, this feature could be set up so each sample will advance to a melt stage 2 cycles after each sample crosses the threshold.


To use this feature the Analysis Settings must be set up (in the Create Run screen) before the run starts, because the analysis settings define the threshold. If the analysis settings are changed after the run has completed or after a site has already advanced to the next stage, the results may not make sense. To setup this feature follow the instructions below.

Defining a Protocol with Advance to Next Stage

1. Click the New Protocol button. The New Protocol dialog box will open.

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Figure 4-80: The New Protocol dialog box.

2. Enter a unique name up to 32 characters (avoid using the following charac-ters: ^ | < > : * ? \ /).

3. Click OK to add the new protocol name to the Protocol Name list.

4. Select Hold from the drop-down stage type menu in Stage 1.

Figure 4-81: The Stage Type drop-down menu and a completed Hold stage.

5. Enter a Hold temperature (acceptable range is 40–98 °C) and the number of seconds the site should remain at that temperature (acceptable range is 3–3200 seconds). An optical reading can not be programmed during a Hold stage.

6. Select 2-Temperature Cycle, 3-Temperature Cycle, 4-Temperature Cycle or 5-Temperature Cycle from the stage type drop-down menu in Stage 2.

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Figure 4-82: Selecting a Temperature Cycle stage.

7. Enter the number of times to repeat the temperature cycle (integers between 1–100).

8. Enter ramp rates, temperatures, times and read information for the stage.

Figure 4-83: A completed three-stage Temperature Cycle protocol stage.

9. Check the box next to Advance to Next Stage.

Figure 4-84: A 3-Temperature Cycle stage with Advance to Next Stage enabled.

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10. Enter the number of cycles to wait after the cycle threshold before advanc-ing to the next stage.

11. Select a channel to monitor for a cycle threshold. Enter Ch1 for the interca-lating dye.

12. Leave the next stage blank so that each site will stop the specified number of cycles after the cycle threshold or select Melt Curve for the intercalating dye so that each site will advance to the melt stage the specified number of cycles after the threshold crossing.

13. Click Save Protocol.

Editing Protocols1. Highlight a protocol to edit in the Protocol Name list and make appropriate

changes to the protocol stage(s).

2. Click the Save Protocol button to implement the changes.

All runs performed using the previous version of the protocol will retain the original settings. The new settings will be applied to all new runs created after the protocol is saved.

If a protocol is modified after it has been assigned to sites for a run that has not yet started (i.e., the run is still in the Create Run screen), click Cancel Run Setup so that the new version of the protocol will be implemented. Never use two versions of a protocol with the same name in a run.

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4.6 The Define Graphs Screen

Figure 4-85: The Define Graphs screen.

Use the buttons and fields in the Define Graphs screen to create and edit graph definitions. All of the graphs saved in the SmartCycler database are listed in the Graph column on the left side of the screen. If no new graphs have been created, the list will display the nine default SmartCycler graphs.

• Temperature — plots the sample temperature.

• FAM — plots channel 1 primary curve fluorescence vs. cycle number.

• Alexa Fluor 532 — plots channel 2 primary curve fluorescence vs. cycle number.

• Cy3 — plots channel 2 primary curve fluorescence vs. cycle number.

• TET — plots channel 2 primary curve fluorescence vs. cycle number.

• Texas Red — plots channel 3 primary curve fluorescence vs. cycle number.

• Alexa Fluor 647 — plots channel 4 primary curve fluorescence vs. cycle number.

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The Define Graphs Screen

• Cy5— plots channel 4 primary curve fluorescence vs. cycle number.

• Standard — plots the log concentration vs. cycle threshold for standards (blue diamonds) and unknowns (red rectangles) for one channel only.

• intcltr_dye — plots channel 1 primary curve fluorescence vs. cycle num-ber for channel 1.

• Melt — plots fluorescence vs. temperature, and also places a red vertical line through the highest peak(s) of the first derivative, and designates the temperature corresponding to the peak as the Tm.

When a graph is selected from the Graph list, the details of the graph setup are displayed in the area to the right of the list.

Note: If a graph that has been associated with a saved run is modified, the next time that run is displayed, the graph will show the new settings.

Creating a New Temperature GraphTemperature graphs plot temperature (in °C) vs. time (in Hr:Min:Sec).

1. Select the New Graph button.

2. Enter a unique graph name.

Figure 4-86: Entering a new temperature graph name.

3. Click OK to add the graph name to the Graph list.

4. Click the Graph Type arrow and select Temperature from the drop-down menu.

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Figure 4-87: The Graph Type drop-down menu.

5. Select the type(s) of temperature data to display (Figure 4-88):

• Sample Temperature — the calculated temperature of the samples.

• Heater Temperature — the measured temperature of the I-CORE heaters.

• Target Temperature — the programmed temperature specified in the protocol stage.

Figure 4-88: The Temperature graph options.

6. Check the box next to Automatically added to new Runs to automatically add this graph to the Views list of every new run.

7. Click Save Graph.

Creating a New Optics GraphOptics graphs display a plot of optical signal (Fluorescence) vs. Cycle or Log Fluorescence vs. Cycle.



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Figure 4-89: The New Graph Name dialog box.

3. Click OK to add the graph to the Graph list.

4. Click the Graph Type arrow and select Optics from the drop-down menu.


5. Select the type(s) of optics data to display (Figure 4-91):

• Channel — select up to four channels to display on a single graph (e.g., check Ch 1 for FAM).

• Show: Primary Curve — displays the growth curve.

• Show: 2nd Derivative — displays the 2nd derivative curve.

• Show: Threshold (Horizontal) — displays the user-defined threshold.

• Show: Threshold Crossings (Vertical) — displays the cycle threshold.

• Axes: Fluorescence vs. Cycle — displays fluorescence signal vs. cycle number

• Axes: Log Fluorescence vs. Cycle — displays log fluorescence vs. cycle number.

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Figure 4-91: The Optics graph options.

6. Check the box next to Automatically added to new Runs to automatically add this graph to the Views list of every new run.

7. Click Save Graph.

If multiple channels are plotted, the data from each channel will be plotted using a different symbol:

Figure 4-92: A graph displaying data from all four channels.

Ch1: plain line

Ch4: diamonds

Ch2: crosses

Ch3: asterisks

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Creating a New Standard Curve GraphA standard curve graph displays a plot of log concentration vs. cycle threshold.



Figure 4-93: Entering a new standard curve graph name.


4. Click the Graph Type arrow and select Standard Curve from the drop-down menu.


5. Select the type(s) of data to display(Figure 4-95):

• Channel — select the desired channel.

• Show — standard points are plotted as blue diamonds and unknown points are plotted as red rectangles.

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Figure 4-95: The Standard Curve graph options.

Creating a New Melt GraphA melt graph plots fluorescence vs. temperature (in °C).



Figure 4-96: Entering a new melt graph name.


4. Click the Graph Type arrow and select Melt from the drop-down menu.

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Figure 4-97: The Graph Type menu.

5. Select the type(s) of data to display(Figure 4-98):

• Melt Curve — select to show the plot of fluorescence vs. temperature.

• 1st Derivative — select to display the 1st derivative of the melt curve.

• Melt Temperature(s) (Tm) — select to display the melt temperature rep-resented by a red vertical line with an annotated value (in °C) at the larg-est peak(s) of the first derivative.

Figure 4-98: The Melt graph options.

Note: Only data from the channel selected in the protocol (Melt stage Optics field) will be graphed. Refer to Figure 4-79 on page 110.

Duplicating a GraphThe Duplicate Graph button can also be used to define a new graph.

1. Highlight a graph name in the Graph list.

2. Click the Duplicate Graph button.

3. Enter a new name in the Duplicate Graph Name dialog box.

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Figure 4-99: The Duplicate Graph Name dialog box.


5. Modify the graph definition.

6. Click the Save Graph button.

Editing GraphsTo edit a graph, click on the graph name in the Graph list to display the graph definition, then modify the graph settings, and click the Save Graph button.

To rename a graph, highlight the graph name in the Graph list, then click the Rename Graph button. Enter a new name in the Rename Graph dialog box, then click the Save Graph button.

To re-order the graph names in the Graph list, highlight a graph name and click the Move To Top button.

Deleting Graphs1. Highlight a graph name in the Graph list.

2. Click the Delete Graph button.

3. Click Yes in the Delete Graph dialog box to delete the graph or No to cancel the action.

Figure 4-100: The Delete Graph dialog box.

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The Stop Run Button

4.7 The Stop Run ButtonUse the Stop Run button to terminate runs or specific sites before they have finished. The button is only active after a run has been started, at other times it is grayed and inactive.

Figure 4-101: The Stop Run button.

1. Click the Stop Run button to open the Select Run(s) or Site(s) to Stop dialog box.

2. Click the arrow to select whether you want to stop Runs or Sites from the drop-down menu.

Figure 4-102: Selecting whether to stop Runs or Sites.

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Figure 4-103: Stopping runs or sites in the Select Run(s) or Site(s) to Stop dialog box.

Stopped sites can be used in a new run immediately even if other sites are in use.

• To stop a run in progress, select Sites from the drop-down menu, then highlight the run name and click the Stop button.

• To stop all runs in progress, click the Stop All button and then click Yes to confirm.

• To stop individual sites, select Sites from the drop-down menu then highlight the sites and click the Stop button.

• To stop all sites, click the Stop All button and then click Yes to confirm.

• Click the Cancel button to close the Stop dialog box without stop-ping runs or sites.

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The Check Status screen

4.8 The Check Status screen

Figure 4-104: The Check Status screen.

The Check Status screen displays the following information about each run that has been started since the software was last opened:

• Run Name

• Status (Running, Done or Stopped)

• Number of Sites

• Protocols

• Time Remaining (Hr:Min:Sec)

Click a run name in the Name column of the Runs Started box to view the Site ID, Cycle Status, Protocol and Status of each site in the selected run.

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4.9 The Maintenance Screen

Figure 4-105: The Maintenance screen.

The Instruments table displays the following information about each processing block currently connected to the computer:

• Instrument identification letter — the designation of each processing block connected to the SmartCycler System: A, B, C, etc.

• Device S/N — the serial number of the processing block.

• Model — the model number of the processing block.

• Internal Temperature — the temperature inside the processing block.

• Site — the site number assigned to each I-CORE module in the process-ing block.

• S/N — the serial number of the I-CORE module for each site.

• Run Starts — the number of runs started in the I-CORE since the last cal-ibration.

• Last Cal — the date the I-CORE was last calibrated.

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The Maintenance Screen

The Messages section displays error messages and warnings. See Chapter 7 “Troubleshooting” for more details on error messages and warnings.

If any of the I-CORE modules have started more than 900 runs since the last calibration, a warning message will be displayed above the Instrument table.

The Maintenance screen also displays SmartCycler Software version, the current user and the date and time the SmartCycler Software was opened (upper right corner of the screen).

Error MessagesThe Messages field at the bottom of the Maintenance screen displays the date and time the software was last started, the software version, and a scrollable list including any error messages or warnings that were detected since the SmartCycler Software was opened. If errors or warnings were detected they are included in the Run Report. A file containing the error and warning messages will also be saved in the Errors folder of the SmartCycler Software folder. The full path to the Errors folder is:

My Computer > (C:) > SmartCycler > Errors

The error files are named with the run name + “.dat”. To open an error file:

3. Right-click on Run Name.

4. Select Open with....

5. Select WordPad.

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Chapter 5: Tutorial: Running SmartCycler Assays

5.1 Using the SmartCycler® Reaction Tubes . . . . . . . . . . . . . . . . . . 131• Handling the Reaction Tubes . . . . . . . . . . . . . . . . . . . . . . . . 131• Using the Tube Puller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133• Using the Tube Rack and Cooling Block. . . . . . . . . . . . . . . 134

5.2 Running a B-Actin Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135• Setting Up the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135• Preparing the Master Mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . 135• Creating the Sample Dilutions . . . . . . . . . . . . . . . . . . . . . . . 136• Opening the SmartCycler Software. . . . . . . . . . . . . . . . . . . 137• Setting Up the SmartCycler Software. . . . . . . . . . . . . . . . . 138• Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

5.3 Applying Quantitative Analysis to the B-Actin Assay . . . . . . . 144• Setting Up a Standard Curve Using Primary Curve

Analysis 144• Setting up a Standard Curve Using 2nd Derivative Curve

Analysis 146• Quantitating Unknowns With an Imported Standard

Curve 148

5.4 Setting Up Melt Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 154• Defining a Melt Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

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• Viewing Intercalating Dye Melt Results . . . . . . . . . . . . . . . 155

5.5 Overview: Running an Assay With an Internal Control . . . . . . 158• Setting up the SmartCycler Software for an Internal

Control 158

5.6 Overview: Running a Multiplex Assay with a Quantitative Internal Control (QIC) 160

• QIC Analysis in a Quantitative Assay. . . . . . . . . . . . . . . . . . 160• QIC Analysis in a Qualitative Assay . . . . . . . . . . . . . . . . . . . 163

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5.1 Using the SmartCycler® Reaction Tubes

Handling the Reaction TubesThe polypropylene SmartCycler reaction tube is designed for optimal thermal and optical characteristics. The optical detection windows are along the bottom edges of the tube, as indicated in Figure 5-1.

Figure 5-1: The SmartCycler 25 μL reaction tube.

Handle the reaction tube by the ribbed upper portion of the tube. Avoid touching the optical detection windows at the bottom edges of the tube and the sides of diamond-shaped area.

Always use filter-barrier tips or filter-barrier gel-loading tips to fill SmartCycler reaction tubes. If you are using a gel-loading tip, hold the tube at eye level and insert the tip approximately halfway into the diamond-shaped area of the tube and dispense the reaction mixture slowly to minimize trapping bubbles.

It is essential that the cap is seated properly in the tube before the reaction tube is closed. When capped correctly, the tube becomes pressurized and the sides of the diamond-shaped area of the reaction tube bulge slightly. When the tube is inserted into the tube slot in the I-CORE® module, this convex shape provides proper contact between the sides of the tube and the heaters to

Cap front

Ribs

Cap back

Opticalexcitation/detection

window

Opticalexcitation/detection

window

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ensure efficient thermal cycling and amplification. If the cap is not completely closed and the tube is not pressurized, amplification may not be as efficient. When closing a reaction tube, it is recommended to put the tube in the Smart-Cycler reaction tube rack, then press down on the cap until you feel a distinct “snap”.

Bubbles in the reaction mixture can interfere with optical measurements. To remove bubbles, centrifuge the tubes for 2–4 seconds in the mini-centrifuge included with the SmartCycler system.

Insert the tube into the modified rotor so that it is as horizontal as possible, as shown in Figure 5-2. Avoid handling the optical detection windows at the tube bottom edges and the film-covered diamond-shaped area.

Figure 5-2: Positioning SmartCycler reaction tubes in the modified rotor.

Insert the filled and capped reaction tube into the I-CORE site slot, press down firmly into place. The reaction tube can be inserted with either the front or back of the cap facing the front of the SmartCycler processing block.

To remove a reaction tube from the I-CORE slot, firmly grasp the sides of the tube and pull straight up. Be careful to not accidentally open the cap of the tube, which could release some of the contents onto the SmartCycler processing block. You many find it easier to remove the tubes using the tube puller included with your SmartCycler System (see “Using the Tube Puller” on page 133).

To remove the reaction mixture from the tube after amplification, centrifuge the tube briefly before the cap is opened. This will force any liquid near the cap down into the diamond shaped area of the tube and help to reduce the possi-

Rotor

Correctly positioned

Not correctlypositioned

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Using the SmartCycler® Reaction Tubes

bility of aerosol contamination. Open the cap carefully. Remove the liquid with a filter-barrier gel-loading tip.

Using the Tube PullerThe tube puller can make it easier to remove reaction tubes from I-CORE modules. Hold the tool perpendicular to the top surface of the I-CORE module, then slide the two prongs around the neck of the reaction tube. If the cover of the adjacent I-CORE module is closed, tilt the tool at a slight angle to fit it between the I-CORE cover and the tube.

Note: Be sure to insert the prongs between the “ribs” on the neck of the reaction tube, not directly under the cap. If the tube puller is placed directly under the cap, the cap will open.

After the tube puller prongs are in place, pull the tube straight up, as shown in Figure 5-3. Do not rock the tube from side to side (i.e., toward either of the adjacent I-CORE modules), and be careful not to pull it at an angle. If additional force is needed, rock the tube gently towards the front of the processing block, then rock it gently toward the back of the processing block.

Figure 5-3: Using the tube puller to remove reaction tubes from an I-CORE module.

Pull straight upSlide prongsbetween ribs

Do not rockside-to-side

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Using the Tube Rack and Cooling BlockThe SmartCycler tube rack keeps the tubes upright in the cooling block slots. In addition to providing support for the tubes, the rack slots are labelled with numbers corresponding to the numbers of the I-CORE modules in the Smart-Cycler processing block, making it easy to ensure the correct tube is inserted into each module. Tube racks can be disinfected by wiping or soaking them in 10% bleach, then rinsing well.

Figure 5-4: The SmartCycler cooling block, shown with a tube rack.

The SmartCycler cooling block provides even, dry cooling for up to 16 reaction tubes for more stable and consistent assay results. Always place a tube rack on top of the cooling block before inserting reaction tubes. Store the cooling block in the refrigerator, so that it’s at the right temperature when needed. The block temperature increases about 10 °C/hour at average room temperature. Place the block in a bucket of ice to keep the temperature of the block constant for a long period of time.

The cooling block provides a steady base for the tube rack to make tube capping easier. If you are capping tubes at the side positions of the rack (e.g., in position 1 or 9), it’s helpful to steady the opposite side of the cooling block to prevent it from tipping sideways when exerting force to snap the caps shut.

To clean the cooling block, wipe or soak it with 70% ethanol, then rinse with distilled water and dry well. To disinfect the cooling block, soak it briefly in a 10% bleach solution (no more than 5 minutes), rinse with copious amounts of distilled water and dry completely.

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Caution: Do not leave the block submerged in bleach longer than the recom-mended time — the metal can corrode.

5.2 Running a B-Actin AssayA five-sample Beta-actin (B-actin) assay, using commercially available reagents, is used to illustrate the setup and execution of a DNA amplification run. This procedure can also be used as a training exercise to become familiar with the SmartCycler System or for confirming the performance of the System.

Setting Up the ReagentsThaw the following reagents and store them on ice throughout the reagent preparation:

• Forward/Reverse primers and probe from the TaqMan Beta-actin control kit (Applied Biosystems, Cat# 401846)

• Human DNA Target, diluted to 150,000 molecules/2 μL (Promega, Cat# G3041).

• OmniMix HS Kit (Cepheid Cat# OMNI1-100N-050)

Preparing the Master MixPrepare enough master mix for six (5 samples + 1 extra)25 μL reactions. Add the reagents in the order listed below to a 1.5 mL eppendorf tube. Briefly vortex and centrifuge each reagent before using it to ensure even distribution. After the PCR beads are added, cap the tube and gently tap or vortex until the beads dissolve — be sure there is no bead residue clinging to the sides of the tube. After adding all reagents, vortex the master mix gently, then place on ice.

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Table 5–1: Master mix set up for B-actin assay.

Creating the Sample Dilutions1. In this experiment, 0, 150, 1,500, 15,000 and 150,000 molecules of human

genomic DNA will be amplified. Dilute the human genomic DNA stock ~ 1.5 x 105 molecules/2 μL, in DEPC treated water to 15,000, 1,500 and 150 mole-cules/2 μL. The sample volume for this experiment is 2 μL, if a larger sample volume per reaction tube is desired, adjust the amount of water in the mas-ter mix so the total volume of the master mix plus sample is 25 μL.

2. Use a filter-barrier pipette tip or filter-barrier gel-loading tip to place 23 μL of the master mix in a 25 μL reaction tube. If you are using a gel- loading tip, insert it about halfway into the diamond-shaped area of the reaction tube and dispense the master mix slowly to minimize trapping bubbles in the dia-mond-shaped area.

Reagent25 μL Reaction

Final Concentration

DEPC Water 93 μL

Forward Primer (3 μM) 15 μL 0.3 μM

Reverse Primer (3 μM) 15 μL 0.3 μM

Probe (2 μM) 15 μL 0.2 μM

OmniMix HS PCR Kit (do not count towards total volume)

3 beads 25 mM Hepes, pH 8.0, 4 mM MgCl2, 200 μM dNTPs, 3U TaKaRa hot start Taq polymerase

Master Mix Volume per 6 reactions (with-out DNA)

138 μL

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3. Add 2 μL of the DNA sample to each tube as follows:

Table 5–2: Sample set up for B-actin assay.

4. Close the tubes completely (see “Handling the Reaction Tubes” on page 131). Place the tubes in the modified rotor and centrifuge 2 to 4 seconds. Remove the reaction tubes from the centrifuge and check for bubbles. If any bubbles remain in the reaction mixture, centrifuge again.

5. Insert each SmartCycler reaction tube into sites 1-5 in the SmartCycler pro-cessing block.

Opening the SmartCycler SoftwareTurn the SmartCycler processing block on (the switch is located on the back of the processing block near the top). If more than one SmartCycler processing block is attached to the computer, turn them on one at a time in the order specified in “Installing Additional SmartCycler Processing Blocks” on page 19.

To open the SmartCycler Software:

1. Double-click the SmartCycler icon on the desktop, or from the Windows Start menu select Programs > Cepheid > Cepheid SmartCycler.


2. The splash screen will appear, and two “A’s” should light up and flash on the top of the SmartCycler processing block (if more than one processing block

SampleDNA Concentration

Master Mix Volume

Sample Volume

Total Reaction Volume

1 0 23 μL 2 μL water 25 μL

2 150 23 μL 2 μL 25 μL

3 1500 23 μL 2 μL 25 μL

4 15,000 23 μL 2 μL 25 μL

5 150,000 23 μL 2 μL 25 μL

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is installed, the first will be “A”, the second “B” and so on), indicating the firmware has downloaded to the processing block.

Note: To close the SmartCycler Software, choose Exit from the User menu, or click the close box in the top right corner of the SmartCycler Software window.

Setting Up the SmartCycler Software1. Click the Define Protocols icon to define the B-actin protocol.

2. Click the New Protocol button to open the New Protocol dialog box. Enter B-actin 2 Step for the new protocol name (a unique name is required) and click OK.

3. Define Stage 1 (the hold stage):

Figure 5-6: The Hold stage for the B-actin 2 Step protocol.

• Select Hold from the drop-down menu of stage types.

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4. Define Stage 2:

Figure 5-7: Defining Stage 2 of the B-actin 2 Step protocol.

5. Click the Save Protocol button.

6. Click the Create Run icon to create a new run. Enter Beta actin in the Run Name field.

Note: A unique run name is required. The software does not allow duplicate run names.

7. Click the arrow in the Dye Set box and select FCTC25 (FCTC represents FAM, Cy3, Texas Red, and Cy5).

8. Click the Add/Remove Sites button to open the Select Protocols and Sites dialog box. The B-actin 2 Step protocol should be highlighted.

9. Highlight sites A1 through A5, then click the right arrow to transfer the selected sites and protocol to the Selections field. Click OK to save the selections.

• Select 2-Temperature Cycle protocol from the drop-down menu.

• Enter 40 in the Repeat field at the top of the Stage 2 box to specify that it should be repeated for 40 cycles

• Enter 95 for Temp and 15 for Secs in the first step.

• Enter 68 for Temp and 30 for Secs in the second step.

• Click in the Optics cell of the second step and select On from the drop-down menu. This sets detection of the fluorescence signal to occur at the end of the second step in each cycle.

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Figure 5-8: Selecting sites for the B-actin assay run.

10. Review the run setup and click Start Run.

Figure 5-9: The B-actin assay run setup.

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Viewing ResultsAfter Start Run is clicked the orange LEDs on the SmartCycler processing block will turn on at sites A1-A5 and the software will switch to the View Results screen (Figure 5-10).


1. Select Analysis Settings from the Views list. Set up the Analysis Settings for FAM as follows:

• Usage — accept the default of Assay for the FAM channel and select Unused for Cy3, TxR and Cy5 channels.

• Bkgnd Sub — accept the default value of ON.

• Bkgnd Min Cycle & Bkgnd Max Cycle — accept the default values of 5 (Min) and 40 (Max).

• Curve Analysis — accept the default of Primary Curve.

• Thresh Setting — accept the default of Manual.

• Manual Thresh Fluor Units — accept the default of 30 (fluorescence units).

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• Boxcar Avg — accept the default value of 0.

• Target — enter B-actin.

2. Click the Update Analysis button to implement changes.

3. Click the Save Run button.

4. Click the Define Graphs icon to define new graphs. Click the New Graph button at the bottom of the screen, then enter FAM Primary with Threshold for the new graph name in the New Graph Name dialog box.

5. Set up the new graph as follows:

Figure 5-11: The FAM Primary with Threshold graph definition.

6. Click the View Results icon and click the Select Graphs button to associate the FAM Primary with Threshold graph with the run.

7. Click Temperature in the Views list to display the real time temperature pro-files for each site (Figure 5-12). This graph displays the temperatures of the reaction mixtures (for more information on default graph types, see “The Define Graphs Screen” on page 114).

• Graph Type — click the arrow and select Optics from the drop-down menu.

• Channel(s) — accept the default, Ch 1.

• Show — select Threshold (Horizontal) and Threshold Crossings (Vertical) and accept the default, Primary Curve.

• Axes — accept the default, Fluores-cence vs. Cycle.

• Click the Save Graph button at the bot-tom of the screen to save the graph.

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Note: Although the temperature data from the individual I-CORE modules start out in phase, they will progressively spread apart during the course of the run. This is normal because the rate at which each I-CORE module can cool varies slightly. The amount of time each sample is held at each target temperature is consistent in all of the I-CORE modules; only the rate of cooling varies slightly and the amplification efficiencies are not affected.

Figure 5-12: Temperature profiles during the B-actin assay.

8. To view temperature profiles for selected sites, click on a color-coded Site ID in the graph legend. Use the [Shift] key to select contiguous sites or the [Ctrl] key to select non-contiguous sites.

9. Select FAM in the Views list to display the growth curves in real time (i.e., the graph of fluorescence signal vs. cycle). If the growth curves are visible above the background signal, select the FAM Primary with Threshold graph. The manual threshold will be marked with a red horizontal line intersecting the Y axis at 30. When a growth curve crosses this threshold, a red vertical line will be drawn through the intersection. The interpolated cycle threshold value (Ct) will be displayed on the graph and in the Results Table (Figure 5-13).

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Figure 5-13: A B-actin assay primary curve with threshold at 30 units and cycle threshold value of 29.98 for 1500 molecules.

10. After the run has finished, the data can be exported or printed (see “Export-ing Data” on page 96 and “Viewing and Printing Reports” on page 99 for more information).

5.3 Applying Quantitative Analysis to the B-Actin Assay

The SmartCycler System can be used to run quantitative assays. This section describes how to apply quantitative analysis to the B-actin assay created in section 5.2.

Setting Up a Standard Curve Using Primary Curve Analysis1. Select Results Table in the Views list. Set up the Results Table table as fol-

lows for each site:

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Applying Quantitative Analysis to the B-Actin Assay

Figure 5-14: Setting up standards in the Results Table.


3. Enter the concentration (i.e., starting molecules of DNA) of each standard. The acceptable range is 0.001 to 1.0E9.

4. Select Analysis Settings from the Views list. Verify that the settings for FAM are as follows:

• Usage — accept the default of Assay.







• Target — enter B-actin.

5. Click the Update Analysis button to implement changes.

6. Select Standard in the Views list.

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Figure 5-15: Viewing a standard curve.

Notes: y = ax+ b, r-squared = c (where a, b, and c are all numbers). a defines the slope, b is the Y intercept and r-squared is the correlation coeffi-cient, which defines how well the standards fit to a straight line.

The Standard graph plots standards (STD) as blue diamonds and unknowns (UNKN) as red squares.

Setting up a Standard Curve Using 2nd Derivative Curve Analysis

Define a new graph to display the 2nd derivative of the growth curve with the threshold, threshold crossing and cycle threshold:

1. Click the Define Graphs icon and highlight the FAM Primary with Threshold graph name.

2. Click the Duplicate Graph button and enter FAM 2nd Deriv in the Duplicate Graph Name dialog box.

3. Check 2nd Derivative under Show and click the Save Graph button (see “Creating a New Temperature Graph” on page 115 for more information).

4. Click the View Results icon and click the Select Graphs button to associate the FAM 2nd Deriv graph with the run.

5. Change the Curve Analysis to 2nd Deriv.

Unknown Standard


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6. Click Analysis Settings in the Views list.

7. Select 2nd Deriv from the Curve Analysis column drop-down menu.

Figure 5-16: Changing Curve Analysis to 2nd Deriv.

8. Click the Update Analysis button.

9. Click FAM 2nd Deriv in the Views list.

Figure 5-17: A growth curve with 2nd derivative curve analysis.

10. Select the Standard graph in the Views list and a new standard curve and linear fit equation will be displayed, representing the cycle thresholds for the 2nd derivative data.

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Figure 5-18: The Standards graph when the 2nd derivative is used to detect the cycle threshold.

Quantitating Unknowns With an Imported Standard CurveIt is possible to quantitate unknown samples with a standard curve from another run by importing the standard curve and Analysis Settings into a run that contains only unknowns (UNKN).

Notes: The Analysis Settings from the standard curve are imported with the standard curve and will temporarily override the Analysis Settings in the current run.

It is not possible to import a standard curve into a run that contains multiple protocols or standards.

1. Before importing a standard curve, note the Analysis Settings (especially Curve Analysis, Thresh Setting and Boxcar Avg) and the “DYE” Ct results for the current run.

2. Click Analysis Settings and Results Table in the Views list to display the set-tings and results for the current run.

Unknown Standard


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Figure 5-19: The Analysis Settings and Results Table.

3. To import the analysis settings and standard curve from a previous run, click the Import Std Curve button, highlight a Run Name from the list of runs that contain valid standard curves and click OK.

Figure 5-20: Imported standard curve results and quantitation of unknowns.

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4. Select Analysis Settings to display how the data for the imported standard curve and current run data are analyzed.

Select Results Table to display the imported standard curve results (high-lighted in yellow) and the current run (UNKN) results.

Click Standard in the Views list to display the imported standard curve and quantitated unknowns.

Notes: Quantitation of UNKNs is displayed in the “DYE” Std/Res column.

The default Standard graph only displays standards for Ch 1. Create a new graph to display standards in other channels.

Figure 5-21: The imported standard curve with quantitated unknowns.

Modifying the Combined Run

It is not possible to view or modify the Analysis Settings for the current run while a standard curve is imported. Click the Remove Std Curve button or save the combined run to make changes while a standard curve is imported.

To make changes to the Analysis Settings or Results Table of the imported standard curve run:

1. Click the Remove Std Curve button.

2. Click the View Another Run button.

Standard (blue)

Imported standard curve

Unknown (red)

Unknowns

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3. Select the run name of the imported standard curve and click OK.

4. Modify the Analysis Settings and click the Update Analysis button.

5. Click the Save Run button.

6. Click the View Another Run button and select the current run name.

7. Click the Import Std Curve button to import the modified Analysis Settings and standard curve.

Saving the Combined Run

1. Click the Save Combined Run button.

2. Enter a new name for the combined run.

Figure 5-22: Entering a new name for a combined run.

3. Click OK.

Note: It is recommended you enter the names of each run in the Notes field for future reference.

Exporting Data From the Combined Run

To export data with an imported standard curve:

1. Click the Save Run button before exporting data.

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Note: The SmartCycler Software does not give a prompt to save changes before printing or exporting. Therefore, it is possible to make changes to the Results Table or Analysis Settings, and immediately print or export the data, then close the run without saving the changes, in which case the data saved in the SmartCycler database will not match the printed or exported data.

2. Click the Export button to display the Export Data dialog box.


3. Select type of data to export:

• Export Optics Data — by default, Primary Curve and 2nd Deriv data are exported for all channels except channels designated as Unused in the analysis settings.

• Export Results Table and Analysis Settings —exports the Results Table and Analysis Settings as they are displayed in the View Results screen.

• Export Melt Data — exports primary and first derivative melt data by default.

• Export Heater Temperature Data — exports temperature data for each site.

4. Select a folder from the Look in drop-down menu or accept the default Export folder.

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Figure 5-24: Selecting a folder and entering a name for an export file.

5. Click Save.

Data are exported as comma-delimited text (.csv) files that can be opened with MS Excel. By default, the file is exported to the Export folder in C: >SmartCycler >Export (a shortcut to this folder is installed on the desktop). If multiple types of data are exported in a single file, the file will display multiple tables of data in one worksheet.

When Export Optics Data is selected, the primary curve data and/or 2nd deriv-ative data for each channel will be exported except channels designated as Unused in Analysis Settings (unless default is set up differently). The exported primary curve and 2nd derivative data will reflect the boxcar average cycles and background subtraction analysis settings. For example, if Bkgnd Sub is ON the background subtracted data will be exported, whereas if the Bkgnd Sub is OFF the raw data is exported.

Figure 5-25: An example of exported optics data.

Dye name

Site ID

2nd Derivative

Displays target name (if entered in Analysis Settings) or repeats dye name

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Printing a Report of the Combined Run

To preview or print a report with the imported standard curve, click the Report button.

5.4 Setting Up Melt Curve AnalysisIntercalating dyes intercalate all double-stranded DNA including primer-dimers so it is necessary to program a melt curve to immediately follow amplification to discriminate between specific and non-specific products. Melt curves provide information about the purity of the amplification product and the melting temperature of the amplified product(s). Melt curves are generated by slowly ramping up the temperature to 95 °C, and as the DNA melts or becomes single-stranded the intercalating dye is released and the fluorescence signal decreases. Analysis of the melt curve by the first derivative can identify the melting temperature (Tm) of the amplified product(s). The Tm of a specific DNA sequence is the temperature at which 50% is in double-stranded form and 50% is in single-stranded form and is dependent on amplicon length and GC content.


The melt curve plots the increasing temperature vs. the measured fluorescence. The SmartCycler Software uses the first derivative to determine the Tm. The top of the first derivative peak corresponds to the Tm. Up to five Tms can be detected per sample so if multiple amplicons are present, and the length and GC content differ significantly, the melt curve will have more than one peak and Tm.

Defining a Melt Protocol1. To set up the cycling protocol for the melt curve, click the Define Protocols

icon and define a protocol using the settings shown in Figure 5-26.

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Setting Up Melt Curve Analysis

Figure 5-26: The b-actin 2-stage protocol.

2. Select Melt Curve from the Stage 3 drop-down menu. Accept the default Start temperature (60 °C), End temperature (95 °C) and ramp rate (0.2 Deg/Sec).

Figure 5-27: Defining a Melt Curve stage after the B-actin thermal cycling stages.

Viewing Intercalating Dye Melt ResultsThe intercalating dye melt results will be displayed in the default Melt graph.

Figure 5-28: Intercalating dye melt curve.

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To define two additional melt graphs:

Creating a First Derivative Graph

The software automatically determines the melting temperature of each sample by analyzing the first derivative of the melt curve.

1. Click the Define Graphs icon and highlight the default Melt graph in the left column

Figure 5-29: Highlighting a graph to duplicate.

2. Click the Duplicate Graph button and enter Melt Deriv in the Graph Name dialog box.

3. Check 1st Derivative and Melt Temperature under Show and click the Save Graph button (see “Creating a New Melt Graph” on page 120 for more infor-mation).

Figure 5-30: Selecting Melt Deriv graph attributes.

4. Click the View Results icon and click the Select Graphs button to associate the Melt Deriv graph with the run.

5. To view the Melt graph in real time select Melt in the Views list.

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Setting Up Melt Curve Analysis

Figure 5-31: Displaying a melt curve and 1st derivative peak.

6. Select Results Table in the Views list and scroll to the right to view the cal-culated Tm(s). The number of Melt Peak columns displayed will depend on how the melt setting defaults have been defined (Setup > System Defaults > Melt Settings). If you have selected to show a maximum of 5 peaks, the Results Table will display columns for Melt Peak 1– Melt Peak 5. Melt Peak 1 will display the melt temperature for the largest peak, Melt Peak 2 will dis-play the second largest peak, etc. Only results for melt peaks that meet the acceptance criteria defined in the melt setting defaults will be displayed in the Results Table. For example, if you have selected to show a maximum of 5 melt peaks but only 1 peak meets the acceptance criteria, only 1 melt tem-perature will be displayed.

Figure 5-32: The reported melt temperatures in the Results Table.

Select Melt in the Views list to display:

Melt curve

1st derivativepeak

Tm

Select Melt Deriv in the Views list to display:

Tm

1st derivativepeak

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5.5 Overview: Running an Assay With an Internal Control

An internal control can be used to validate an assay. Acceptable performance of an internal control confirms that the reagents are functional and the calculated cycle threshold for the unknown is valid. When an internal control is used, an acceptable cycle threshold range for the internal control is designated prior to starting the run. This range should be narrow enough to differentiate positive and negative results. If the internal control crosses the threshold outside of the specified acceptable range, then the analysis of the unknown is not valid and ND (not determined) will be reported in the Results Table for all channels for that sample.

Multiplexing is required to utilize the Internal Control SmartCycler Software feature. A two-target multiplex assay will be used to demonstrate the use of an internal control. The example below illustrates the use of an Internal Control channel (TxR) and an Assay channel (FAM).

Setting up the SmartCycler Software for an Internal Control1. Select Analysis Settings from the Views list.

2. Set up the FAM row as follows:








• Target — enter the target name.

3. Click the Update Analysis button to implement the changes.

4. Set up the TxR row as follows:

• Usage — select Internal Control.

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Overview: Running an Assay With an Internal Control






• Valid Min & Valid Max — enter a valid cycle range (e.g., 27 to 30).




6. To interpret results for the internal control and unknown, click Results Table in the Views list.

Figure 5-33: The Analysis Settings and Results Table for an internal control assay.

Note: Analysis settings are applied to the raw data, but do not change the raw data. Analysis settings can be changed before, during or after the run is finished.

Valid Internal Control Ct

Invalid Internal Control Ct

Valid Ct range for Internal Control

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5.6 Overview: Running a Multiplex Assay with aQuantitative Internal Control (QIC)

A quantitative internal control (QIC) is used to correct for differences in assay performance due to normal site-to-site and run-to-run variations. It also corrects for differences due to variability in reagents and any minor inhibition due to sample components. The quantitative internal control is added at a constant concentration to the target reaction mixture that includes an unknown sample. The cycle threshold of the unknown is divided by the cycle threshold of the QIC. The resultant ratio is used for qualitative and quantitative analysis of the unknown. For example, if the cycle threshold of the designated QIC in the TxR dye channel is 30 and the cycle threshold of the unknown sample in the FAM dye channel is 31, then the ratio of 31/30 (1.033) is displayed in the Results Table and used for analysis. Only the channel designated as the QIC will display a cycle threshold value in the Results Table.

When optimizing a multiplex assay that includes a quantitative internal control, it is important to completely characterize all assay components. The quanti-tative internal control should have the same PCR efficiency as the test targets run in the sample mixture since small changes in efficiency can translate into large changes in total product formed (Innis, PCR Strategies, 1995). It is important to run the QIC at a concentration that will produce reliable and reproducible cycle thresholds so that the corrections to the unknown samples are accurate.

QIC Analysis in a Quantitative AssayWhen using a quantitative internal control for quantitative analysis, the standard curve for the target will be generated by plotting the cycle threshold ratio vs. log concentration. The concentration of the QIC target is constant in all samples and the assay target is serially diluted.

The following example demonstrates the use of a QIC using a model two-target multiplex. TxR is the QIC target and FAM is the assay target. The standard concentrations are: 10, 100, 1000 and 10,000 copies.

Setting up the Analysis Settings

1. Select Analysis Settings from the Views list.

2. Set up the FAM row as follows:


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Overview: Running a Multiplex Assay with a Quantitative Internal Control (QIC)









4. Set up the TxR row as follows:

• Usage — select QIC.






• Valid Min & Valid Max — enter a valid cycle range (e.g., 29 to 30).




Setting Up a Standard Curve

1. Click the Results Table in the Views list.

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Figure 5-34: The View Results screen for an assay that includes a quantitative internal control.


3. Enter the concentration (i.e., the starting copy number of DNA) of each standard in the FAM Std/Res column.

4. If the QIC channel crosses the threshold outside of the specified acceptable range, then the analysis of the unknown is not valid and ND (not deter-mined) will be reported in the Results Table for all channels for that sample.

If the QIC and Assay channels cross the threshold within the specified acceptable range, PASS is reported in the TxR Std/Res column and quantita-tion of the UNKN sites is reported in the FAM Std/Res column.

The TxR DYE Ct column reports the Ct value for each site.

The FAM Ct columns report the ratio of FAM DYE Ct to TxR DYE Ct.

5. Click on Standard in the Views list to display the plot of the FAM Ct to TxR Ct ratio vs. log concentration of the standards.

TxR (QIC) CtRatio of FAM Ct to TxR Ct

Quantitation of unknown

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Overview: Running a Multiplex Assay with a Quantitative Internal Control (QIC)

Figure 5-35: The Standard graph of log concentration vs. ratio for a duplex QIC, showing the standards (blue dots) and the unknowns (red squares).

QIC Analysis in a Qualitative AssayThe QIC function can also be used in a qualitative assay. See “QIC Analysis in a Quantitative Assay” on page 160 for the proper Analysis Settings.

To view the results for the QIC and Assay channels, select Results Table.

Figure 5-36: The Results Table for QIC analysis in a qualitative assay.

Select UNKN from the Sample Type drop-down menu for each site.

If the QIC crosses the threshold outside of the specified acceptable range, the analysis of the unknown is not valid and FAIL will be reported in the Results Table for all channels for that sample.

Unknown

Standard

Ratio of FAM (Assay) Ct to TxR (QIC) Ct

No Ctdetermined

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If the QIC crosses the threshold within the acceptable range, but the Assay channels do not report a Ct, PASS is reported for QIC and NEG is reported for the Assay channels in the Results Table for that sample.

If the QIC crosses the threshold within the specified acceptable range and the Assay channel reports a Ct, a POS result is reported in the Results Table for that sample.

The QIC DYE Ct column reports the Ct value for each site.

The Assay DYE Ct columns report the ratio of Assay DYE Ct to QIC DYE Ct.

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Chapter 6: Maintenance

6.1 Removing and Replacing I-CORE® Modules . . . . . . . . . . . . . . . . 167

6.2 I-CORE Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

6.3 Disinfecting the SmartCycler System and I-CORE Modules . . . . . 173

6.4 Disinfecting the Tube Rack and Cooling Block . . . . . . . . . . . . . 174

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6.1 Removing and Replacing I-CORE® Modules

Warning:The following procedure must only be performed after power has been removed from the instrument. This will avoid the risk of electric shock and possible damage to the equipment.

1. Turn off the processing block and unplug the power cord.

2. Remove the 6 phillips-head screws that retain the top cover (2 screws in front, 2 screws in back and 1 on each side, see Figure 6-1).

Figure 6-1: Removing the phillips-head screws from the top cover.

Carefully lift up the top cover and swing it down to the side of the processing block. The cable assemblies may have to be gently pulled out a little bit to allow the cover to rest on the benchtop (see Figure 6-2).

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Figure 6-2: Opening the top cover.

3. Remove the top 2 phillips-head screws and loosen the bottom 2 screws on the back cover (i.e., the side of the processing block closest to the On/Off switch). This will create a gap between the back cover and the instrument’s internal frame that will allow you to remove the metal shields that cover the I-CORE modules.

4. Remove both of the metal shields (see Figure 6-3A and Figure 6-3B). The back cover may need to be pulled back slightly to free the shield.

Warning:The edges of the metal shields are quite sharp. Handle very carefully!

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Figure 6-3: Removing the shields that cover the I-CORE modules.

5. Carefully remove the I-CORE module from the base, using both hands and employing a slight rocking motion from front to back, not from side to side. Be careful not to snag or damage the two flat ribbon cables on each side of the I-CORE assembly (see Figure 6-4).

A

B

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Figure 6-4: Loosening and removing the old I-CORE module.

6. Remove the new I-CORE module from its electrostatic discharge (ESD) box. Save the box — you’ll need it to package the old I-CORE module for shipping.

7. Insert the new I-CORE module into the base, positioning it so that the key on the card socket aligns with the corresponding notch in the module’s cir-cuit board (see Figure 6-5). The two rows of I-CORE modules are inserted 180° from each other.

Figure 6-5: Detail of notch in the I-CORE module’s card and the key in the card socket.

Notch

Key

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8. Reinstall the smaller metal shield first, then position the larger shield over the I-CORE modules. The cable assemblies must be positioned between the leftmost I-CORE modules and the sheet metal wall and should not be pinched by the metal shield; the cable should be routed through the cutouts in the front and rear left corners of the shield. You may need to slightly move the I-CORE modules side-to-side or front-to-back to get the shield to drop-down in place. Make sure the holes in the metal shield (2 in the front and 2 in the back) align with the threaded buttons on the front and back housing flanges. Re-tighten the 2 lower phillips-head screws on the back cover, then insert and tighten the 2 upper screws.

Figure 6-6: Checking the cable routing through the metal shield.

9. After installing the shield, double-check that the cable assemblies are posi-tioned as shown in Figure 6-6 before you swing the cover assembly back over the top of the instrument. This ensures that the cable assemblies will be in the cutaway area of the metal shield and prevents them from being pinched. Also check that all 16 gray rubber I-CORE sealing gaskets are cor-rectly in place (Figure 6-7).

Cable assembly

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Figure 6-7: The I-CORE sealing gaskets in place over the I-CORE modules.

10. Re-attach the top cover with the 6 screws you previously removed.

11. Package the old I-CORE module in the ESD bag that contained the new I-CORE module and place it in the box that contained the replacement I-CORE module. Fill out the Compliance Certificate completely, then insert it under the shipping label on the front of the box, making sure that the RMA number and shipping address are both clearly visible.

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I-CORE Calibration

6.2 I-CORE CalibrationIf any of the I-CORE modules have had more than 900 runs started since their last calibration, a warning message will appear when the SmartCycler Software is started. The Maintenance screen displays the number of run starts for each I-CORE module in each processing block currently connected to the computer.

Figure 6-8: The Maintenance screen.

6.3 Disinfecting the SmartCycler System and I-CORE Modules

If a SmartCycler processing block or an I-CORE module needs to be returned to the Service Department for repair, it must be disinfected prior to shipment using the following procedure. This same procedure can be used as a safety measure if this equipment is routinely exposed to biohazard materials.

Wipe all outside surfaces of the SmartCycler instrument and/or the I-CORE module with a 10% bleach solution. After 10 minutes, wipe all the same surfaces with a 70% alcohol solution.

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Caution:Do not allow any of the bleach or alcohol solution to drip or seep into the tube slot of the I-CORE module. The alcohol solution can damage the lenses of the I-CORE module’s optical blocks, which can adversely effect performance.

6.4 Disinfecting the Tube Rack and Cooling BlockThe SmartCycler tube racks can be disinfected by wiping or soaking them in 10% bleach, then rinsing well.

If you need to clean the cooling block, wipe or soak it with 70% ethanol, then rinse with distilled water and dry well. To disinfect the cooling block, soak it briefly in a 10% bleach solution (no more than 5 minutes), rinse with copious amounts of distilled water and dry completely.

Caution:Do not leave the block submerged in bleach longer than the recom-mended time — the metal coating can corrode.

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Chapter 7: Troubleshooting

Processing Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Optics Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Temperature Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Standard Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Melt Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Results Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Warning Messages and Error Codes. . . . . . . . . . . . . . . . . . . . . . . 193

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Symptom Cause Solution

1.Message: “No Devices! There are no instruments available, so some functions will not be allowed.”

a. The processing block is turned off.

• For data analysis only: select OK and proceed. Only the following screens will be available: View Results, Define Graphs, Define Protocols, and Maintenance.

• Select OK. Close software. Turn on processing block. Open software.

b. USB cable not connected.

• Check USB cable connection.

2. Message: “At least one I-CORE module in Instrument X has more than 900 run starts. Please contact customer service to schedule calibration, which is recommended every 1000 run starts or 12 months, whichever comes first.”

a. 900 runs have been started on at least one I-CORE module since the last calibration. Check Maintenance screen to see how many runs have been started on each I-CORE module.

• Calibration is recommended either annually or every 1000 runs. The Service Coordinator will contact you to arrange for the processing block to be sent to the Service Department for calibration.

3. The processing block does not light up and does not display 0/1.

a. The processing block does not have power.

• Check that power cord is completely plugged in.

• Try plugging processing block into a different power outlet.

• Check that power switch is turned on.

• Check power cord for breaks or other damage.

• Check the circuit breaker in the back of the processing block. Press the square red button to reset.

• If more than one processing block is daisy-chained together, make sure the processing block closest to the computer is turned on.

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4. Add new hardware error message says that user does not have privileges to install devices.

a. Processing block is being installed for the first time and user does not have administrator rights.

• Log in to Windows as an Administrator. Click Start select Shut Down..., Log off, OK. At the Windows login prompt enter Administrator as the user and leave the password field blank and click OK.

5. After opening software, the processing block continues to display 0/1 rather than A/A or B/B, etc.

OrMessage: “Problem with communications, attempting to recover. Please disconnect and re-connect USB cable(s) to computer and make sure USB cable(s) are connected to processing block(s) Recovery may take several minutes.”

OrMaintenance screen displays a flashing yellow icon indicating there is a problem with data collection, and that the instrument is trying to recover.

a. Communication between the computer and the processing block is disconnected.

• Check that the USB cable is securely plugged into the processing block and the computer. (See Chapter 2 “Installation”.)

• Close the SmartCycler Software and turn off the processing block (leave computer on). Unplug the USB cable from the computer and re-insert it. Turn on the processing block. Wait for processing block to display 0/1. Open the software.

• Check the USB cable for breaks or other damage.

• Check USB driver(s): right-click My Computer. Select the Hardware tab in the Properties dialog, Click the Device Manager button. One Cepheid SmartCycler USB driver should appear for each processing block.

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Computer


1.Message: Database cannot be connected. Please check for valid database and that the SQL Service server manager is running.

2. Computer asks for new run name even though the current run name is not a duplicate run name.

3. Define Protocols or Define Graphs screen is empty.

4. After Start Run, software does not switch screens from Create Run to View Results.

5. Stop Run or Check Status screens are grayed out.

6. Cannot stop run or close software.

a. The computer name has been changed.

• For If new computer name is required, close all Windows applications and SmartCycler Software. Insert SmartCycler version 2.0 CD into computer. It will start up automatically. Click Next, Yes, Custom. Un-check SmartCycler files and Shrink Database Log. Check Install MSDE. Click Next, Yes for “Are you sure” Keep default settings. Click Next. Wait for uninstall and re-install of MSDE. Click Finish for “Yes, I want to restart my computer now”.

b. The MSSQL Server has been stopped.

• Double-click MSSQL Server icon at bottom right corner of

taskbar. Select Start/Continue. Check Auto-start service when operating system starts. Close SQL Server Service Manager.

7. The Import Std Curve button is grayed out

a. Current run has multiple protocols.

• It is not possible to import a standard curve into a run that has multiple protocols.

b. Current run has sites designated as STD.

• Select Results Table and change Sample Type to UNKN for all sites.

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8. SmartCycler Software does not respond while the processing block is in use. No data.

a. Windows power scheme was altered to allow System standby, Turn off monitor or Turn off hard disks.

• Select Start > Settings > Control Panel > Power Options. Check that: Power Schemes=SmartCycler DO NOT ALTER, System standby= Never, Turn off monitor =Never, Turn off hard disks=Never.

b. User logged into Windows 2000/XP with a user name having Power Schemes set to allow System standby, Turn off monitor, or Turn off hard disks.

• For every new user, set Power Schemes to Always On, System standby= Never, Turn off monitor =Never, Turn off hard disks=Never.

9. Safe Mode displayed. a. Power interference. • Start in Normal mode. Shut down and restart if computer is unresponsive or very slow.

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10.SmartCycler Software is not responding.

a. Data communication interrupted or lost.

• Unplug and re-plug USB cable to establish USB recovery. USB recovery can take up to 5 minutes.

• Remove any additional equipment connected to the SmartCycler computer via a USB cable (e.g., USB connected printer or zip drive).

• Make sure that SmartCycler computer and processing block are plugged into a surge protector.

• Check Norton Anti-virus settings. Open Norton Anti-Virus, select Options > Auto-Protect, Un-check Start Auto-Protect when Windows starts up. Select Scan system files at startup. Click OK.

b. Another software program is running while SmartCycler Software is in use.

• Do not open or use other programs or use the internet while SmartCycler Software is in use.

• Do not use any equipment that is connected to the SmartCycler computer via a USB cable.

• Click [Ctrl] + [Alt] + [Del] to see if a program is not responding.

Computer


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11.Message: Old database version or invalid file name appears when retrieving a run.

a. Retrieving an exported (.CSV) file or a database (.DAT) file.

• Check that the file being retrieved is an archived run file with .RUN as the extension.

b. Restoring an exported (.CSV) or an archive run (.RUN) file.

• Check that the file being restored is a backup database file with .DAT as the extension.

c. Retrieving an archived run file or restoring a database backup file from a network location or CD.

• Copy the archived run or database backup file from the network location or CD to the local drive (C drive). Windows 2000 requires advanced security settings to retrieve and restore to a network location. For more information call Technical Support.

12.Error encountered during archive or backup.

a. Performing archiving or backup to network location.

• Archive or backup to local drive (C drive) and copy to network. For more information call Technical Support.

Optics Graphs


1.No data available… displayed at bottom of graph window.

a. Graph has been altered or is not defined correctly.

• Go to Define Graphs. Verify that Graph Type=Optics for an optics graph or Graph Type = Temperature for a temperature graph. Make appropriate changes so that graph name and graph definition are the same and save graph.

b. First 2 cycles have not yet completed.

• Wait for additional cycles to be completed.

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2. Software stops collecting data before end of run.

a. Protocol exceeded time limit of 14,400 sec. (4 hours).

• Decrease time of one or more steps of protocol.

3. Ct (vertical line) does not intersect the threshold (horizontal line) at the same point as growth curve intersects the threshold.

a. Curve Analysis=2nd Deriv and does not match graph defined to show only the Primary Curve.

• Change Curve Analysis = Primary Curve.

• In Define Graphs, create a new graph to show: Primary Curve, 2nd Deriv., Threshold and Threshold Crossing.

• Select a graph that shows 2nd Deriv.

4. Inconsistent or irreproducible results. Replicate samples do not show replicate results.

a. “Sticky” targets or amplicon.

• Try SmartCycler Additive Reagent or other PCR enhancer. See “Getting Started” application note.

b. Sticky enzyme. • Try a different enzyme.

c. Very low copy numbers, at limit of detection for assay.

• Increase sample concentration.

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5. No data displayed in optical (FAM, etc.) graph or temperature graph of View Results screen.

a. Computer date or time was changed while the SmartCycler Software was running.

• Do not change computer date/time while SmartCycler Software is open.

• If the date/time is changed while a run is in progress the communications between the software and the processing block will be lost and data loss will result.

b. Optics were not turned On in protocol.

• In the Protocols view, check that Optics=On at appropriate temperature step in cycle. If Optics=Off, go to Define Protocols. Choose protocol, make appropriate changes and save protocol. Repeat experiment.

c. Graph definition is incorrect.

• Verify graph definition in Define Graphs.

d. User logged out of Windows while a run was in progress.

• Logging off terminates any open programs. Any unsaved data will be lost if a user logs out of Windows while the SmartCycler Software is open.

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6. No Ct value or vertical line displayed on optical graph.

a. Automatic Thresh Setting applied 4 cycles after Auto Max Cycle.

• Select Thresh Setting = Manual.

• Wait for all sites to finish running.

b. Threshold setting (horizontal line) is too high.

• If Thresh Setting = Automatic, decrease Auto Thresh #SD's.

• If Thresh Setting = Manual, decrease Manual Thresh Fluor Units.

c. 2nd deriv peak begins in last few cycles of run. 2nd deriv requires 3 more cycles after the 2nd Deriv peak to calculate Ct.

• Change Curve Analysis to Primary Curve.

• Repeat with additional cycles in protocol.

d. Graph definition does not include Threshold.

• Highlight graph name in Define Graphs screen and select Threshold Crossing under Show.

7. Ct (vertical line) does not intersect threshold (horizontal line) at peak of 2nd Deriv.

a. Curve Analysis= Primary Curve and graph is defined to show 2nd Deriv.

• Change Curve Analysis = 2nd Deriv.

• In Define Graphs, change graph to show Primary Curve, Threshold and Threshold Crossing.

• Select a graph that shows only Primary Curve and Threshold.

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8. Growth curve abruptly decreases fluorescence or becomes flat.

a. Fluorescence is too high. Status= Warning see Maintenance screen (Warning: Fluorescence Signal Too High).

• Turn Bkgnd Sub off to view raw fluorescence. If signal is >500 units at cycle 1 or increases above 1000 units, decrease dye or probe concentration. Check purity of probe, high concentration of free dye will increase background fluorescence.

• Decrease fluorescent dye or probe concentration. Reduce final intercalating dye concentration to between 0.125X and 0.5X. Note: Ethidium bromide cannot be used as an intercalating dye in the SmartCycler system.

b. If assay uses an intercalating dye, target concentration may be too high. Note: Ethidium bromide cannot be used as an intercalating dye in the SmartCycler system.

• Dilute sample.

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9. No real time growth curve (primary curve) and no visible bands on gel.

a. Error in master mix preparation or protocol.

• Check master mix calculations and check protocol in Define Protocols screen.

b. Protocol needs to be optimized.

• See “Getting Started” application note and test annealing temperatures and times.

c. Sample component inhibiting PCR.

• Dilute sample or modify extraction procedure.

d. Excess fluorescent dye is inhibiting PCR.

• Decrease fluorescent dye concentration. Serially dilute fluorescent dye and include a positive control without dye. Run samples on gel and compare dye samples to the positive control without dye to check for inhibition.

e. Degraded PCR reagent.

• Repeat with fresh reagents.

f. I-CORE heater failure. • Check temperature cycles in temperature graph for specific site. Refer to “Temperature Graphs” on page 189.

g. Damaged reaction tube.

• Check reaction tube in specific site for tube leakage. Contact Technical Support.

10.Jagged or saw-tooth curve

a. Y-axis=Log fluorescence rather than Fluorescence.

• Open Define Graphs. Highlight graph name. Check box for Fluorescence vs. Cycle and save graph. Return to View Results.

b. Very low fluorescent signal.

• Optimize fluorescent dye or probe concentration. Refer to “Getting Started” application note.

c. Small scale of Y-axis magnifies display of background noise.

• Re-scale graph axis.

• If possible, display with other sites that have growth curves with higher endpoint fluorescence.

d. A problem with the I-CORE module.

• Contact Technical Support.

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11.Growth curve decreases to negative fluorescence or appears abnormal.

a. A sharp increase or decrease in fluorescence in the first few cycles.

• Turn Bkgnd Sub off to view raw signal. Set the Bkgnd Min Cycle to one cycle after the initial increase or decrease and turn Bkgnd Sub back on.

• See “Background Subtraction” on page 29.

b. A threshold crossing was not detected.

• Decrease the Manual Thresh Fluor Units value or Auto Thresh #SDs value until a threshold crossing is detected or alter the Bkgnd Min and Bkgnd Max settings.

12.Very low fluorescence signal.

a. Low copy number of template (target).

• Concentrate or add more sample volume.

b. High background signal. Set Bkgnd Subto OFF to view background. The max. valid fluorescent signal is approximately 1000 fluorescence units. Background >600 limits the dynamic range of the growth curve.

• Decrease dye or probe concentration. Check purity of probe, high concentration of free dye will increase background fluorescence.

• Repeat with fresh reagents.

c. Degraded PCR reagent.

• Avoid freeze/thaw of reagents and light exposure to fluorescent dye or probe. Store according to manufacturer's recommendations.

d. Sample component inhibiting PCR.

• Dilute sample.

• Modify extraction procedure.

e. Damaged reaction tube.

• Check reaction tube for proper closure or leakage.

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13.The growth curve for one site looks abnormal and all other growth curves look normal.

a. I-CORE heater or fan is not functioning properly.

• Check temperature graph profile for site. If profile is abnormal, please contact Technical Support.

b. Sample component inhibiting PCR reaction.

• Dilute sample.

• Modify extraction procedure.

c. There is an air bubble in reaction tube.

• Repeat run and ensure that complete centrifugation removes all air bubbles.

d. Sample volume is too high or too low.

• Fill 25 μL tube with 25 μL water and verify that diamond shaped area of reaction tube is completely filled (small meniscus OK).

e. Reaction tube is leaking or damaged.

• Repeat the run with a new tube. If reaction tube is leaking or damaged, please contact Technical Support and provide lot number of reaction tubes.

Temperature Graphs


1.Thermal cycles do not progress. Processing block appears to pause at a particular step in protocol.

a. Temperature setting is not within acceptable range of 40–98°C.

Note: lowest achievable temperature depends on ambient and internal temperature of the processing block. See “Installing the SmartCycler® System” on page 17.

• In View Results screen, select Protocols from Views list. Programmed temperatures must be within the acceptable range (40–98°C). If a programmed temperature is outside of acceptable range, modify and save the protocol in Define Protocols.

• Contact Technical Support.

2. A site shows temperature spikes or abnormal temperature profiles.

a. I-CORE module needs to be replaced.

• Please contact Technical Support.

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3. No temperature profiles displayed in temperature graph or optical curves displayed in optics graph.

a. Temperature graph is incorrectly defined.

• Go to Define Graphs, check Graph Type = Temperature. If not, make changes and save graph.

a. Loss of communication during the run.

• Unplug and re-plug the USB cable to initiate USB recovery. USB recovery can take up to 5 minutes.

Standard Curves


1.No data available... a. Graph not defined correctly.

• In Define Graphs screen, select graph, check Graph Type = Standard Curve and Channel = Ch [#]. Save graph.

b. No standard concentrations have been entered.

• Select Results Table from View list, In Sample Type column, change UNKN to STD. In “DYE” Std/Res column, enter numeric value for standard concentration. After at least 2 standards have been entered, click on any cell in a different column then click the Update Analysis button.

c. Threshold set too high so Ct values not detected for standards.

• For Manual Thresh Setting decrease fluorescence unit value in the Manual Thresh Fluor Units column. For Automatic Thresh Setting, decrease value in Auto Thresh # SD’s column.

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2. Standard curve is plotted as a vertical line or single point.

a. Incorrect values were entered for standards in the DYE Std/Res column.

• Check standard concentrations: lowest standard = highest Ct; highest standard = lowest Ct.

b. The same Ct values were calculated for 2 different standard concentrations.

• For the standard that does not correspond to the standard curve, change the sample type from STD to UNKN.

• Include Additive Reagent to improve reproducibility and possibly sensitivity, see “Getting Started” application note.

3. Import Std Curve button grayed out.

a. Run includes multiple protocols.

• It is not possible to import a standard curve into a run that contains multiple protocols.

4. “DYE” Std/Res=ND although Ct is not zero and a red data point is plotted for this unknown on the graph.

a. Unknown value is more than 1/2 log above the highest standard or 1/2 log below the lowest standard.

• Extend dynamic range of standards to include unknown values.

• Dilute sample or add less sample volume.

• Concentrate sample or add more sample volume to quantitate unknowns within standard curve dynamic range.

Standard Curves


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Melt Curves


1.Large amount of non-specific product amplified.

a. Excess primer. • Decrease primer concentration.

b. Degraded intercalating dye. Note: Ethidium bromide cannot be used as an intercalating dye in the SmartCycler system.

• Make fresh dilution of intercalating dye for each experiment. Protect from light (e.g., wrap in foil). Store according to vendor’s recommendations.

• Order new intercalating dye.

c. Reaction starting before thermal cycling begins.

• Use hot start polymerase.

• Keep reaction mixture chilled until tubes are inserted into the processing block.

d. Temperature of optical read step in protocol too low.

• 2-step protocol: increase anneal temperature.

• 3-step protocol: program optical read during extension step.

• Add an extra step to the protocol to read the optics at a temperature higher than the Tm of primer-dimers.

e. Excess intercalating dye. Note: Ethidium bromide cannot be used as an intercalating dye in the SmartCycler system.

• Decrease intercalating dye concentration. Test 1:2 serial dilutions of intercalating dye from 0.5X to 0.125X.

f. Primer design not optimal.

• Check the sequence for homology.

g. Excessive hold times. • Decrease hold times: denaturation, anneal and/or extension.

2. Melt curve stopped before the End temperature was reached.

a. Ramp rate too fast. Melts that run from high to low temperatures require a slower ramp rate.

• Decrease the Deg/Sec ramp value to 0.1 or 0.2 deg/sec.

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Warning Messages and Error CodesThe SmartCycler Software reports errors and warnings in the Check Status screen and in the View Results screen in the Results Table. If a deviation or error has occurred during the run, the Status column will display Warning or Error. If no error has occurred, the Status column will display OK.

The warning and/or error details are recorded in the Messages section of the Maintenance screen while a run is in progress. After the run is complete the errors or warnings are recorded at the end of the Run Report and in the Errors folder. Click Report at the bottom of the View Results screen to view the Run Report.

If the software has remained open since the run was finished, the Maintenance screen will display error details in chronological order. The day/month/year/time and block/site number are recorded for each error.

If the software has been closed since the run was finished, the details will no longer be displayed in the Maintenance screen but are stored in an Errors folder. To view errors from previous runs: Open My Computer > C: > Smart-Cycler > Errors. A list of all runs in which an error or warning was recorded will be displayed. Select the file titled by run name and open in WordPad by right clicking, then selecting WordPad. Error details are listed in chronological order.

Results Table


1.Changes in Analysis Settings are not reflected in Results Table results.

a. The Update Analysis button was not clicked after changes were made.

• Click the Update Analysis button.

Exporting Data


1.Export dialog box remains on screen.

a. Temperature data too large to export.

• Compact temperature data by saving run before exporting.

• Always click the Save Run button before exporting.

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The warning messages and error codes are:

Warning/Error Cause Solution Code

Optical Detec-tor Offset Error During Measurement

Lid is not completely closed. Outside light source affects detector's ability to estab-lish a baseline.

Ensure that lid is com-pletely closed.

3072

Temperature Out of Range Error

The actual temperature is more than +/- 0.5 °C from the target temperature within the range of 60 °C to 95 °C.

Contact Technical Sup-port for possible I-CORE module replacement.

3073

Oven Temper-ature Differ-ential Error

The temperature difference between Oven A and B was greater than 5 °C. Possible I-CORE heater failure.

Contact Technical Sup-port for possible I-CORE module replacement.

3074

Warning: Site May Be Empty

Temperature ramped down more quickly than expected. The I-CORE site may be missing a tube.

Check that the I-CORE site contains a reaction tube.

3076

Internal Tem-perature Error

The processing block inter-nal temperature is out of range (<15 °C or >50 °C). The processing block may be located in direct sunlight, near a window, or directly below a heating vent.

Place the SmartCycler System away from direct sunlight and if possible, in a cooler area of the lab.

3078

Warning: Fluo-rescence Sig-nal Too High

Fluorescent signal too high.

Possible I-CORE LED or pho-todiode detector problem.

Decrease/titrate fluores-cent dye or probe con-centration. Check that the I-CORE lid is com-pletely closed. Contact Technical Support for possible I-CORE module replacement.

3079

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Image Corrup-tion Error

A communication error has occurred.

Close the software and turn off the processing block. Wait 5 seconds. Turn on processing block and open software. If error repeats, contact Technical Support for further assistance.

3081

No EE prom data at boot.

Processing block may need calibration.

Contact Technical Sup-port for further assis-tance.

3082

Unable to Reach Tem-perature (block/site#)

Processing block was unable to reach target temperature within 5 minutes for default ramp rate or 15 minutes for user-defined ramp rate (i.e., lowest temperature was below processing block internal temperature).

Check that target tem-perature is within acceptable range (40–98 °C) and above the internal temperature of the processing block. See Internal Temperature Error.

3083

Optics Test Failure

SmartCycler Software detected a problem with optics in an I-CORE module.

Check site for blockage.

Contact Technical Sup-port.

3084

Run-Data-Buffer Overrun

Processing block and com-puter lost communication. The data buffer length has been exceeded. Some data has been lost.

See Processing Block symptom 5 in Trouble-shooting section.

3086

Optical Detec-tor Offset Adjustment at Start Up

Lid is not completely closed. Not able to establish a base-line at start up.

Ensure that lid is com-pletely closed.

3087

Warning: Tem-perature Out of Range

Actual temperature greater than +/- 0.5 °C from the tar-get temperature; outside the 60–98 °C range.

Check if protocol tem-peratures are below 98 °C and 5–10 °C above internal temperature of the instrument.

4097

Warning/Error Cause Solution Code

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Appendix A: User Administration

A.1 Definitions• User Administration— a system designed to preserve the integrity of

runs, protocols, and graphs by implementing password and user-defined access.

• Right — ability to use defined software functions. Example: the ability to delete runs.

• User — an individual given access to specific SmartCycler Software functions.

• Backdoor Password — enables users to access all user administration functions when locked out of the system. The password will be provided through technical support and will be valid for one day.

A.2 User AdministrationUser administration is an optional feature that requires all users to log in to gain access to the SmartCycler software. Each user is configured to have rights to specific software features. If user administration is activated, the system can be set up so that users can access only their own protocols, graphs and runs.

Note: If no users have been defined the user management system will remain inactive.

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Logging In and Logging Out of the SystemUsers can log in and out of the system without closing the software. Therefore, the user can log out of the system while a run is in progress to prevent other users from having access to the run.

To log in while the software is open:

1. Select Login from the User menu. The SmartCycler Login dialog will open.

Figure A-1: The SmartCycler Login dialog box.

2. Enter User Name and Password.

3. Click OK.

Note: The currently logged in user’s ID is displayed on the Maintenance screen.

To log out:

1. Select Logout from the User menu to log out.

Adding or Modifying a User To activate the user management system or modify an existing user password or rights:

1. Select User Administration from the Setup menu to open the User Adminis-tration dialog and click the Add/Modify User tab.

2. Enter a unique user name in the Enter New User field.

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User Administration

3. (Optional) Enter a full name in the Enter Full Name field. The software defaults to the user name if left blank (32 character maximum). The double quote character (“) is not allowed.

Figure A-2: The Add/Modify User tab of the User Administration dialog.

4. Enter a unique password (0–10 characters) in the Enter Password field. A blank password is acceptable. Passwords are case sensitive and displayed in an encrypted asterisk format.

5. Confirm the password in the Confirm Password field.

6. Click Apply.

Note: At least one user must be a system administrator. Only system adminis-trators can add or modify users.

Defining User Access RightsTo define user access rights:

1. Select the Configure User tab of the User Administration dialog.

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2. Click Select User to highlight and select a User Name from the drop-down menu.

3. Un-check individual Access Rights to prohibit access to specific software functions (all access rights are checked by default).

Note: If the System Administration box is checked, the user will have access to all features, including user management, archive and retrieve, and optical calibration.

Figure A-3: The Configure User tab of the User Administration dialog.

4. Click the Apply button to implement changes.

Note: One user must have access to System Administration to ensure access to User Management. Contact technical support for the “backdoor” password if you are locked out of the system.

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User Administration

Deleting a UserTo delete individual users:

1. Click the Delete User tab of the User Administration dialog.

2. Select a user name from the Select User to Delete drop-down menu.

3. Click the Delete button.

Figure A-4: Deleting a user.

4. Click Yes to delete the user or No to cancel the action.

Figure A-5: The Delete User dialog box.

Changing a PasswordTo change your password:

1. Log in to the SmartCycler Software

2. Select Change Password from the User menu.

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3. Enter Old and New Password (0-10 characters). Blank passwords are allowed.

Figure A-6: The Change Password dialog.

4. Enter New Password again to confirm.

5. Click OK. If the new password was valid, and the confirmation password matches the new password, a dialog will confirm the password was changed. Click OK to confirm that password was successfully changed.

Figure A-7: Confirmation that a password has successfully been changed.

If the new password was not valid, or the confirmation password did not match the new password, an invalid password warning will appear (Figure A-8). Click OK and try again.

Figure A-8: The invalid password warning.

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Appendix B: Optical Calibration

B.1 Optical CalibrationOptical calibration is the data stored in each I-CORE module that converts detected signal to fluorescent units for the four channels. Each I-CORE module can store 6 separate optical calibrations (“Dye Sets”): four factory calibrations and two user-defined calibrations. User-defined calibrations can be created for fluorescent dyes, probes, and assays that are different from the factory calibra-tions supplied with the SmartCycler System. Please read the “User-defined Optical Calibration” application note before performing an optical calibration. This application note discusses the critical characteristics of fluorescent dyes and provides guidelines for selecting and preparing calibration solutions. After the calibration solutions have been prepared, follow the procedure described below to create a new user-defined dye set.

The four factory calibrated dye sets are FCTC25 (FAM, Cy3, Texas Red, Cy5, 25 μL tubes), FATA25 (FAM, Alexa Fluor 532, Texas Red, Alexa Fluor 647, 25 μL tubes), Intcltr_dye (intercalating dye, 25 μL tubes) and FTTC25 (FAM, TET, Texas Red, Cy5, 25 μl tubes).


To access the optical calibration functions, a SmartCycler processing block must be attached and turned on before selecting Tools > Optical Calibration.

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The functions and features of optical calibration are performed in the four major sections and seven buttons located across the bottom of the window. The buttons have the following functions:

• Select Sites — opens the Select Sites dialog box to select sites to cali-brate or verify.

• Reset to Default — resets the Dye Set Definition, Dyes, and Excitation/Emission Mapping fields to the default settings for the selected dye set.

• Debug Calibration — starts calibration without limits (the data will not be saved to the I-CORE module). Debug Calibration is intended for trouble-shooting calibrations that do not pass.

• Start Calibration — starts calibration.

• Start Verification — starts verification.

• Report — opens the Optical Calibration Report dialog box.

• Exit — closes the Optical Calibration window.

B.2 Optical Calibration Setup1. Turn on the SmartCycler processing block and open the SmartCycler Software.

Note: Make sure that all Analysis Settings, protocols and graphs have been saved prior to performing optical calibration because the SmartCycler Software must be restarted after the optical calibration process has finished.

2. Select Optical Calibration from the Tools menu. The software will open the Optical Calibration window.

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Optical Calibration Setup

Figure B-1: The Optical Calibration window.

Note: If the SmartCycler Software user administration feature has been activated, the user must have optical calibration rights to continue. See Appendix A “User Administration” on page 203 for details.

Calibration Setup InformationEnter calibration setup information in the Operator/Serial Number fields:

1. If user management has been activated, the User Name field will display the name of the user who is logged in. If user management has not been acti-vated, this field will display Unknown.

2. The serial number of the SmartCycler processing block is automatically entered in the Unit Serial # field.

Figure B-2: Operator/Serial Number section of the Optical Calibration window.

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Dye Set Definition 1. Click Dye Set Location and select Dye Set 5 or Dye Set 6 from the drop-

down menu.

Important: The first available dye set location must be used for each site. Dye sets following empty dye set locations will not be available in the Create Run screen.

Figure B-3: Dye Set Definition section of the Optical Calibration window.

The first 4 dye sets are factory dye sets that are calibrated by Cepheid and cannot be altered. If a factory dye set is selected from the drop-down menu the error message shown in Figure B-4 will be displayed.

Figure B-4: Factory calibration dye set error message.

2. Click in the Dye Set Name field and enter a unique name (up to 11 alphanu-meric characters). The name cannot be the same as one of the factory cali-brated dye sets — if it is, an error message will appear (Figure B-5).

Figure B-5: Factory Dye Set Name error message.

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Optical Calibration Setup

Note: You may find it useful to reference the tube size in the name.

3. Enter the temperature that the sites will maintain to stabilize temperature or to denature a sample before the optical read in the Hold Temp field. The acceptable range is 40 °C to 98 °C. The default is 60 °C.

Note: If dye-oligos are used for calibration, set the hold temperature to the same temperature as the optical read step in the thermal cycling protocol. If Molecular Beacons or FRET probes are used for calibration, set the hold temperature to 95 °C and set the read temp to the same temperature that will be used during the optical read in the thermal cycling protocol.

4. Enter the temperature (acceptable range is 40 °C to 98 °C) that the sites will maintain just before the optical read in the Read Temp field. Typically, the same temperature will be used during the optical read in the thermal cycling protocol. The default is 60 °C.

5. Select the appropriate tube size in the Tube Size field (select 25 μL or 100 μL, only one tube size per dye set).

6. Enter dye lot number or the current date in the Dye Set Lot# field.

7. Enter the time (seconds) to hold the I-CORE modules at the Hold Temp in the Hold Time field. The default is 10 seconds.

8. Enter the length of time (seconds) to hold the I-CORE modules at the Read Temp before the optical read in the Read Time field. The default is 10 seconds.

Dyes1. Enter the dye name (up to 7 characters) for each channel in the Name col-

umn. Enter unused or empty in the Name field for the channel(s) that will not be calibrated. If this field is left blank an error message will appear,.

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Figure B-6: Dyes section of the Optical Calibration window with all channels used (left) and with channels 2–4 designated as unused (right).

Figure B-7: The error message that appears when a Name field is left blank in the Dyes section.

2. Enter the dye concentration in the Concentrations column.

Figure B-8: Entering dye concentrations in the Dyes section.

Note: Refer to the “Optical Calibration” application note for information on selecting appropriate dye concentrations. Assign dyes to optical channels in order of excitation wavelength from shortest to longest to ensure proper calibration (i.e., Ch1 = shortest wavelength (FAM) and Ch4 = longest wavelength (Cy5).

Excitation/Emission Mapping1. Enter excitation and emission mapping information for each channel.

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Optical Calibration Procedure

Figure B-9: Excitation and Emission Mapping fields.

• For excitation and emission properties of dyes, refer to the package insert or request this information from the supplier.

• For channels designated as unused or empty, set the excitation and emis-sion mapping to the default ranges. If an unused or empty channel has a range different than defaults, the calibration can be inaccurate.

• When using probes, arrange the dye order so that the excitation wave-length follows the shortest-to-longest wavelength convention. The cali-bration may not pass if the mapping does not follow this convention.

2. Click the Select Sites button at the bottom of the screen. A new window will open and the available sites will be listed in the Selections column. De-select sites that will not be calibrated by highlighting them and clicking the left arrow to transfer them to the Sites column. The Select Sites button is only available if an editable Dye Set is selected, i.e., dye sets 5 and 6.

3. Click OK.

B.3 Optical Calibration Procedure1. Centrifuge all of the tubes to remove any bubbles. Centrifuge again if the

tubes are jarred, dropped, removed from a site or if bubbles are present in the diamond shaped area.

Note: Do not insert the tubes used for calibration into a site more than two times. If a site fails calibration or verification, prepare another reaction tube filled with calibration solution.

Do not calibrate with the same dye in more than one channel.

2. Click the Start Calibration button at the bottom of the screen.

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Figure B-10: Optical calibration procedure.

7. The buffer reading value will be subtracted from each calibration solution reading. After the optical read, the measured fluorescence will be equal to 1000 fluorescent units

Note: If the calibration is aborted or fails, the SmartCycler Software will restart and calibration will default to previously stored data for each I-CORE module. The current calibration will not be saved.

8. If the following message appears, the gain normalized signal (GNS) or signal to background ratio (S/B) does not meet the minimum specification.

3. Click Proceed to continue with calibration.

4. When prompted, place the tubes containing the buffer in the selected sites. Click OK.

5. When prompted, place the tubes containing the dye into the selected sites. Click OK.

6. Insert the tubes containing the remaining dyes when prompted.

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Optical Calibration Procedure

Figure B-11: Calibration error.

The GNS must be above 2000 at a gain setting of 3. If this value is below 2000 the software will give an error message and the calibration will be aborted. Try increasing the dye concentration.

The S/B ratio must be at least 10 to proceed with the calibration process. Several factors including high background signal, low probe concentration or weak dye signal may influence the S/B value. To increase the S/B ratio, try increasing the dye concentration or changing the buffer composition to decrease the buffer reading.

Note: If a calibration reading is outside the valid optical read range, an error message will appear (Figure B-12). If an optical read value is too low, increase dye concentration. If the optical read value is too high, decrease the dye concentration. Refer to the “Optical Calibration” application note for details on selecting appropriate dye concentrations.

Figure B-12: FAM optical read value outside specified range.

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B.4 VerificationAfter calibration has completed (Figure B-13), the user will be prompted to start the verification process. It is recommended to always perform verification after calibration. During verification, the calibrated buffer and dye readings are compared to the acceptance criteria stored in a system file. The acceptance criteria are ±30 fluorescent units for buffer, ±50 fluorescent units for cross-talk and ±200 fluorescent units for each calibration solution.

Figure B-13: Calibration completed message.

• Click Yes to proceed with the verification procedure (see below).

• Click No to save the valid calibration data without verification and shut down the SmartCycler Software.

• Click Cancel to end calibration without saving the data, and shut down the SmartCycler Software.

Verification ProcedureDo not insert the SmartCycler reaction tubes into a site more than two times. If a site fails calibration or verification, prepare another reaction tube filled with dye solution.

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Verification

Figure B-14: The verification procedure.

Figure B-15: The Verification Completed dialog and the Restart Required dialog.

Measured fluorescence of buffer and cross-talk must be 0 ±100 units to pass verification. Measured fluorescence of calibration solution must be 1000 units ± 200 units to pass verification. If the optical read does not fall within the 800–1200 fluorescence unit range, an error message will appear (Figure B-16). Possible causes include a difference in the calibration solution or the site drifted from previous calibration.

1. When prompted, place the tubes con-taining the buffer in the selected sites. Click OK.

2. When prompted, place the tubes con-taining the dye in the selected sites. Click OK.

3. Continue to add tubes until all the cali-brated solutions have been verified.

4. The Verification Completed dialog box will appear when the verification has completed successfully. Click OK.

5. The Restart Required dialog box will appear immediately after the verification process has completed. Click OK to restart the Smart Cycler Software.

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Figure B-16: Verification error message.

B.5 Optical Calibration ReportsAn optical calibration report can be reviewed and printed from the Optical Calibration window.

1. Open the SmartCycler Software and select the Tools> Optical Calibration to open the Optical Calibration window.

2. Click the Report button at the bottom of the Optical Calibration window.

Figure B-17: Setting up an optical calibration report.

Opticalcalibrationdata

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Optical Calibration Reports

3. Ensure that a Unit (processing block) is selected, then select the optical cal-ibration dye set and sites to include in the report from the Sites list (e.g., OpCal5_900639_A2 for Dye Set 5, unit 900639 and site A2 — refer to Figure B-17).

4. The following error message will appear if a unit (processing block) is not selected.

Figure B-18: Optical calibration report error message.

5. If a unit and corresponding sites have been selected, the optical calibration report will be displayed (Figure B-19).

Figure B-19: An optical calibration report.

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B.6 Verification ReportsAn optical verification report can be reviewed and printed from the Optical Calibration window.

1. Open the SmartCycler Software and select the Tools> Optical Calibration to open the Optical Calibration window.

2. Click the Report button at the bottom of the Optical Calibration window, then select the verified dye set and sites to include in the report from the Sites list (e.g., Verify5_900639 for Dye Set 5, unit 900639).

Figure B-20: Generating a verification report.

3. All sites that were verified for the selected dye set will be shown in the veri-fication report.

Verification data

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Verification Reports

Figure B-21: A verification report.

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218 D1819 Rev D

Appendix C: Windows 2000 and Windows XP withthe SmartCycler Software

Cautions to Avoid Data Loss:

Do not change the date or time while the SmartCycler Software is open. If the date or time is changed while a run is in progress all data will be lost. Changing the date or time interrupts communications between the SmartCycler Software and the SmartCycler processing block, no warning message will appear and the communications cannot be restored.

If new users are created, all power settings must be set to Never for each new user. Data will be lost if the system turns off or goes into hiber-nation during a run. Windows 2000: select Start > Settings > Control Panel > Power Options. Set Power Schemes to Always On and verify that Turn Off Monitor, Turn off hard disks, and System Standby are set to Never. Windows XP: select Control Panel > Performance and Mainte-nance > Power Options. Verify that Turn Off Monitor, Turn off hard disks, and System Standby are set to Never. For laptop computers, make sure that Plugged in and Running on batteries are set to Never.

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Appendix C: Windows 2000 and Windows XP with the SmartCycler Software

Do not log off Windows while a run is in progress. If a user logs off Windows, all open programs will be terminated and data for runs in progress will be lost.

C.1 Logging on to WindowsWindows 2000 and Windows XP require all users to log on at startup. For your convenience, a user with administrative rights has been set up at the factory and will automatically log in at startup. For Windows 2000, the user name is Administrator and the password field is left blank; for Windows XP the user name is Cepheid and the password is left blank.

Note: Auto-login is automatically disabled if the computer is connected to a network.

C.2 Performing Database Functions (Archiving and Retrieving)

Database functions such as archiving and retrieving runs and backing up and restoring the database must be performed to or from the local drive (C drive). To store archive and backup files on a network, copy the run or database file from the local drive (C drive) to the network drive or CD.

For more information please call Technical Support at (888) 838-3222.

C.3 Desktop SettingsWindows enables users to have a personal My Documents folder and Desktop settings. However, logging into Windows with a user name other than Adminis-trator can impact the SmartCycler Software because the factory settings will be altered. If new users are created make sure that shared desktop icons (i.e., the SmartCycler icon) and folders (i.e., SmartCycler Export folder, Errors folder and Backup folder) are placed in C: > Documents and Settings> All Users > Desktop. Each individual desktop can be found in C: > Documents and Settings > User> Desktop (where User refers to each defined user).

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Firewalls

Note: If new users are created, all power settings must be set to Never for each new user. Data will be lost if the system turns off or goes into hiber-nation during a run. Windows 2000: select Start > Settings > Control Panel > Power Options. Set Power Schemes to Always On and verify that Turn Off Monitor, Turn off hard disks, and System Standby are set to Never. Windows XP: select Control Panel > Performance and Mainte-nance > Power Options. Verify that Turn Off Monitor, Turn off hard disks, and System Standby are set to Never. For laptop computers, make sure that Plugged in and Running on batteries are set to Never.

C.4 FirewallsA firewall is software that forms a protective boundary that monitors and restricts information that travels between the computer and a network or the internet. In Windows XP, Windows Firewall is turned on by default.

We recommend not installing any third party firewalls onto the Smart Cycler computer, and that you leave Windows Firewall turned on.

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Appendix C: Windows 2000 and Windows XP with the SmartCycler Software

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Appendix D: SmartCycler Technical Specifications

General SpecificationsDimensions:12”w x 12”l x 10”h

Weight:22 lbs per 16-site instrument

Power requirements: 100–240 VAC, 50–60 Hz

Wattage:350 Watts

Thermal Performance ParametersHeating ramp rates (max.): 10 °C/sec from 50 °C to 95 °C

Cooling ramp rates (max.): 2.5 °C/sec from 95 °C to 50 °C

Temperature duration accuracy: ± 1.0 sec from programmed time

Temperature accuracy: ± 0.5 °C from 60 °C to 95 °C

Melt curve programmable ramp rates: 0.1 °C/sec to 1.0 °C/sec

Environmental ParametersOperating conditions: 15 °C to 35 °C

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Appendix D: SmartCycler Technical Specifications

Operating conditions required forMaximum thermal ramp rates: 20 °C to 25 °C

Relative humidity: 10 to 80%, non-condensing

Altitude maximum: 1500 m

Storage conditions: -20 °C to +60 °C

Reaction Site OpticsDye detection limit: <2 nM for FAM/Cy3/TET/Texas Red/Cy5

Dye Channel Characterization

Channel 1 2 3 4

Excitation(nm)

450–495 500–550 565–590 630–650

Emission(nm)

510–527 565–590 606–650 670–750

SimplexDyes

FAM, inter-calating dyea

a.Ethidium bromide cannot be used as an intercalating dye in the SmartCycler system.

Cy3, TETAlexa Fluor 532

Texas Red Cy5Alexa Fluor 647

MultiplexDyes

FAM Cy3, TETAlexa Fluor 532

Texas Red Cy5Alexa Fluor 647

Quenchersb

b.It is not possible to use a TET/TAMRA probe on the SmartCycler System because both TET and TAMRA emit signal in the same channel (channel 2). If use of a FAM/TAMRA probe is required, please contact Technical Support to obtain a special calibration.



BHQ, Eclipse,DABCYL


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Appendix E: SmartCycler Software Release Notes

Improvements in Release 2.0

General

• Windows XP — now supported (in addition to Windows 2000).

• MSDE and JRE — Microsoft SQL Server Desktop Engine and Java Runtime Environment are updated to newer versions

View Results

• Delete Run(s) — no longer available while run(s) in progress to minimize database access issues.

Define Protocol

• Advance to Next Stage — Viewing other runs no longer interferes with an in-progress run that uses the Advance to Next Stage feature.

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Known Limitations and Clarifications

USB Communications

• Attempting to start a run using sites on a unit that is not communicating will cause an error and shut down the application. Do not start a run if there is a blinking caution icon on the Maintenance button (indicating that USB communication has been lost).

• Melt data may be corrupted if USB communication has been lost or recovered while the melt stage is in process.

Create Run

• Switching dye sets resets all run setup information. Make sure that the correct dye set is selected before additional run information is entered.

• Graph changes are not used by the first run created after those changes are made. When creating a run after making graph changes, click Cancel Run Setup and re-create the run setup.

• The Run Setup Report may show the old “DYE” Std Conc column head-ings if the Target name in the run setup was changed after the setup was copied from another run.

Check Status

• The Time Remaining for runs using 2-temperature protocols with user-defined ramp rates may be incorrect. For example, if the 2-temperature protocol uses different ramp rates for the two temperatures, the esti-mated Time Remaining may be inaccurate.

• The Cycle Status on Check Status screen is sometimes displayed as 0/0. If the number of cycles is required it can be obtained from the View Results screen in the optics graph for each individual run.

View Results

• If Analysis Settings are changed without clicking Update Analysis, the Run Report will display new analysis settings but the sample results will be based on previous analysis settings.

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Protocols

• Melt stages using 0.1 °C/sec ramp rates may not report melt temperatures correctly. It is recommended to use ramp rates of at least 0.2 °C/sec for melt stages.

• Melt protocols using 0.5 to 1.0 °C/sec ramp rates that start at high tem-peratures and decrease to lower temperatures may not achieve the target End Temp. When using this type of melt protocol decrease the End Temp. by 2–3 °C.

• Data will only be collected for the first 4 hours of a run even though a protocol longer than 4 hours can be defined.

Graphing

• Scrolling, zooming, and re-scaling of graphs of a run in progress will reset to the auto-formatted scale each time data is collected from the Smart-Cycler processing block(s). These operations should be performed after the run is complete.

• Optics graphs only report cycles with an optics read step. Thus, if a proto-col has cycling stages without optical reads and also cycling stages with optical reads, the cycles reported are only for those containing an optics read step.

• Graph data for a transposed standard curve is not exported. If exported graph data is needed, it should be exported prior to transposing.

Standard Curves

• Switching a site's Sample Type from STD to either UNKN or UNUSED will erase the standard's concentration. Always check standard concentration entries after switching Sample Types.

• Entry of standard concentrations between 0 and 0.001 will revert to 0.00 without a warning message. Only enter standard concentrations within the valid range of 0.001 to 1E9.

Archive and Backup File Names

• Archive and backup files cannot be generated for file names that contain the following characters: * : | < > ? / \ Do not use any of these characters in an archive or backup file name.

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228 D1819 Rev D

Index

Aaccess options 59add/remove sites button 72Advance to Next Stage feature 110Alexa Fluor 532, definition of 27Alexa Fluor 647, definition of 27analysis settings

assigning channel as internal control or QIC 79

Auto Max Cycle column 83Auto Min Cycle column 83Auto Thresh # SD’s column 83background subtraction 79Bkgnd Max Cycle column 80Bkgnd Min Cycle column 79Bkgnd Sub column 79boxcar average 84changing settings 58custom defaults 58Dye Name column 79exporting settings 97, 152implementing changes 78Run-based Auto threshold

setting 82selecting 78–81

analysis settings, continuedSite-based Auto threshold

setting 83target column 81Usage column 79Valid Max Cycle column 84Valid Min Cycle column 84

archiving runs 64assay channel, definition of 27assays

model B-actin assays 135–137multiplex 158using a QIC 160–163using an IC 158–159

assigning sites and protocols 72–73automatic backup 59automatic threshold setting

Run-based Auto 82Site-based Auto 83

Bbackdoor password 197background subtraction

calculation of 30–35definition of 27

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Index

background subtraction, continuedsetting 79setting Bkgnd Max Cycle 80setting Bkgnd Min Cycle 79theory of 30turning off 79turning on 79

background, definition of 27background, exporting data 98, 153backing up the database 66B-actin assay 135–137

defining the protocol 138displaying growth curve 143–144master mix preparation 135–136melt curve protocol 154–155reagents 135run setup 139–140sample dilutions 136standard curve 144–148with cycle threshold analysis 135–144with melt curve analysis 154–157with quantitative analysis 144–148

Bkgnd Max Cycle column 80Bkgnd Min Cycle column 79Bkgnd Sub column 79bkgnd sub column 79boxcar average 84

definition of 27

Ccabling diagrams 18–21calibration

I-CORE modules 126log 126optical 13, 203–217status 127thermal 13

Cancel Run Setup button 74changing default analysis settings 58changing passwords 55

channelsassigning as internal control or QIC 79data collection from all 12, 108definition of 27excitation and emission ranges

10–11, 224selecting appropriate channel for

dyes 224symbols in multi-channel graphs 118usage settings 79see also optical system

Check Status screen 125closing the software 19color key for graphs 90Compact Database command 69Compare Run button 78comparing runs 78, 96computer, connections to 18–19control channel, definition of 27cooling block 134–135copying run settings 75Create Run screen 70–75

Cancel Run Setup button 74copying from another run 75

creatinggraphs 114runs 70–75

Ct, see cycle thresholdcurve analysis, selecting primary or

2nd derivative 80Cy3, definition of 27Cy5, definition of 27cycle threshold (Ct) 35–37

definition of 27displayed in graphs 143displayed in Results Table 143in B-actin assay 135–144setting a valid range 84specifying parameters 81–84standard curve graphs 119

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Index

Ddaisy-chaining processing blocks 19–

22data

displaying graphs 90exported file locations 98, 153exporting 96–99exporting background 98, 153exporting optics data 96–99exporting run data 49from all channels 12, 108graphing selected sites 90printing graphs 93rescaling graphs 91saving 77setting optical read 108

databasecompacting 69complete backup 66complete restore 68

defaultsanalysis settings 58export settings 61graphs 114melt settings 59, 63

Define Graphs screen 114–122creating new graphs 116–122deleting graphs 122duplicating graphs 121editing graphs 122naming graphs 115, 116, 120

Define Protocols screen 104–113Advance to Next Stage feature 110deleting protocols 105duplicating protocols 106editing protocols 113saving protocols 108

definingnew melt graphs 120

defining, continuednew optics graphs 116new standard curve graphs 119protocols 106–113

Delete Another Run button 44Delete Run(s) button 44deleting

graphs 122protocols 105runs 77users 201

desktop, Windows 2000/XP 220dye channels, see channels 27dye set

creating a new dye set 203definition of 28Dye Name column 79function 71

DYE Std/Res columns 87

Eediting graphs 122editing protocols 113enabling automatic backup 59entering sample IDs 73error codes 193–195error messages 193–195export settings 61exporting

analysis settings 97, 152background data 98, 153data 77data from combined run 103, 151exported data file locations 98, 153graph data 94optics data 96–99Results Table 97, 152run data 49setting defaults 61

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Index

FFAM, definition of 28FATA25 dye set 72firewalls (software) 221FTTR25 dye set 72

Ggeneral system defaults 58generating a specimen report 56generating run log 56graph data, exporting 94graphs

adding to runs 95adding to Views lists 95automatically adding to new runs 116color key to sites 90creating 116–122default 114defining 45, 114–122, 142deleting 122display options 91displaying in View Results screen

90–95displaying selected site data 90duplicating 121editing 122enlarging display area 91exporting data 94key to symbols used 118melt curve 156melt peaks 121, 156, 157naming 115, 116, 120optics 116quantitation 119rescaling 91saving as a JPEG 93selecting 95standard curves 119temperature 115

graphs, continuedtypes 117–121viewing multiple 90

growth curveB-actin assay 144definition of 28

HHelp menu 70hold stages (protocols) 106, 111

IIC

assay example 158assigning a channel as 79cycle threshold display 89defining valid range 84setting valid cycle threshold range

84I-CORE modules

calibration log 126calibration> 173components 10disinfecting 173error messages 127inserting and removing reaction

tubes 132number of run starts 126optical system 10out-of-phase temperature data 143safety information 1–6serial numbers 126warnings 127

importing a standard curve 78, 148exporting data 103, 151modifying 150printing a report 103saving 102, 151

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Index

installing additional processing blocks 19–22

installing SmartCycler System 15–22intcltr_dye dye set 72intercalating dyes, appropriate dye

channel 72internal control, see ICinternal temperature 126

LLEDs (optical system) 10limiting access

to graphs 59to protocols 59to runs 59

logging in to Windows 220logging into SmartCycler Software 55logging out of SmartCycler Software 55Logs menu 56

MMaintenance screen 126–127

calibration status 127display of errors 127display of warnings 127serial number information 126

manual threshold setting 81master mix (for B-actin assay) 135melt curves

B-actin assay example 154–157creating 109defining protocol 154–155definition of 28graphs 120, 156melt peaks 121, 157peaks in first derivative 121

melt curves, continuedpeaks in negative first derivative 154stages (protocols) 109

melt peaks 157graphing 121

Melt peaks column (Results Table) 90melt settings, default 59, 63melt temperature (Tm) 121

definition of 29menus

Help 70Logs 56Setup 57Tools 64

modifyingcombined runs 150imported standard curves 150

Move to Top button 106multiplex assays 158

key to graph symbols 118

Nnaming

graphs 115, 116, 120runs 71

Oonline help guide 70opening the software 19optical calibration 13, 203–217optical channels, see channelsoptical data, viewing 42optical reads 108optics

displaying threshold 117exporting data 96–99graphs 116

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Index

Pperforming a backup automatically 59Print Preview dialog 99printing

combined run reports 103graphs 93run reports 99–100run setup parameters 74

processing blocksconnecting to computer 17daisy-chaining 19–22installing additional 19–22internal temperature 126serial numbers 126

Protocol Name list 104protocols 104–113

adding to runs 74defining 38, 106–113definition of 28deleting 105displaying in View Results screen 90duplicating 106editing 113hold stages 106, 111hold times 105–106item in Views lists 90melt curve stages 109naming, illegal characters 106, 111optical reads in 108renaming 106re-ordering Protocol Name list 106saving 106setting optical read times 108stages 29, 104–105temperature cycle stages 107, 111temperature range 105–106viewing site assignments 85

QQIC

assigning to a channel 79cycle threshold display 89defining valid range 84definition of 28qualitative assay example 163quantitative assay example

160–162setting valid cycle threshold

range 84quantitation graphs

standard curve 147standard default graph 115

quantitative analysis, B-actin assay example 144–148

quantitative internal control, see QIC

Rramp (temperature) 29ramp rate, in melt curve protocols 109reaction tubes 12–13, 131–135

and cooling block 134and tube puller 133and tube rack 134centrifuging 132filling and capping 131handling 131removing used samples 132

Remove Std Curve button 150Report button 77reports

Print Preview dialog 99printing results/analysis 48, 99printing run setup 74

rescaling graphs 91reset zoom 91resizing panels 53restoring a backup of the database 68

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Index

Results Table 85–90DYE Std/Res columns 87exporting 97, 152exporting data 97, 152implementing changes 78Melt Peaks column 90Protocol column 85Sample ID column 85Sample Type column 86Site ID column 85viewing 47

retrieving runs 65run log 56runs

adding and removing graphs 95adding sites and protocols 74archiving 64comparing 44, 78, 96creating 39, 70–75definition of 28deleting saved runs 44editing protocols 113exporting data 49, 96–99illegal characters in names 71plotting standard curves 119printing graphs 93printing reports 99–100printing setup parameters 74retrieving 65run log 56saving changes 43, 76setting optical read times 108software, setting up 38–40viewing saved runs 44

Ssafety information 1–6Sample ID column 85sample IDs, entering 73Sample Type column 86

Save Run button 76saving

combined runs 102, 151graphs as JPEGs 93imported standard curves 102, 151protocols 106runs 76

second derivative, definition of 28Select Graphs button 77Select Graphs button 95Select Graphs dialog 95Select Protocols and Sites dialog 72selecting graphs 95selecting protocols and sites 72–73selecting system defaults 57serial numbers 126setting a backup reminder 59setting a manual threshold 81setting a threshold 81–83setting default analysis settings 58setting system defaults 57, 58setting up default export settings 61setting up default melt settings 59, 63setting valid melt temperature range 64Setup menu 57

user administration 57sites

adding to runs 74assigning sample types 86color code in graphs 90definition of 29entering sample ID 85hiding/displaying in graphs 90serial numbers 126see also Results Table

sliding dividers 53SmartCycler Software

background subtraction 30closing 19, 23cycle threshold 35–37defining software terms 27–29

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Index

SmartCycler Software, continuedicons 54menus 55–70opening 19, 22, 137overview of screen functions 53–54reinstalling 23runs 28screens 54sliding dividers 53user administration 197–202user interface 53–55version number 127

SmartCycler Systemconnecting to computer 18–22heating and cooling 10I-CORE modules 9–12installing 15–22optical calibration 13reaction tubes 12–13safety information 1–6technical specifications 223–224temperature range 105–111theory of operation 7–13thermal calibration 13

software, see SmartCycler Softwaresorting runs

by date 77by run name 77by status. 77by user 77

specimen report 56stages, definition of 29

see also melt curvesstandard curve graphs

cycle threshold 119standard curve 119

standard curves 47, 119, 147assigning sample types 47, 86B-actin assay example 144–148importing 78, 148r-squared 119

standard curves, continuedstandard default graph 115symbols used 119

standardsdefinition of 29entering values 86, 87importing 148

steps, definition of 29see also protocol stages

Stop Run button 123symbols used in manual 1system defaults

general 58setting 57

Ttechnical specifications 223–224

channel characterization 224dimensions/weight 223environmental parameters 223power requirements 223reaction site optics 224thermal performance parameters 223

temperatureacceptable range 105entering in protocols 106, 107, 111graphs 115ramp rates 223ramp, definition of 29

temperature profiles, viewing 42TET, definition of 29Texas Red, definition of 29theory of operation 7–13Thresh Setting column 37, 81

selecting manual or automatic 80threshold

definition of 29displayed in graphs 117setting manual or automatic 37specifying parameters 81–84

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Index

Tm see melt temperatureTools menu 64tube puller 133tube racks 134

Uunknowns

assigning sample type 86quantitating 86, 87

Update Analysis button 78Usage column 79USB connections 18–22user administration 58, 197–202

adding/modifying user 198defining user access rights 199definitions 197deleting users 201logging in/out 198passwords 201

User menu 55using custom analysis settings 58

VValid Max Cycle column 84Valid Min Cycle column 84View Another Run button 44, 77View Results screen

analysis settings 78–81Compare Run button 44, 78Delete Run(s) button 44, 77displaying graphs 90–95displaying protocols 90Export button 77Import Std Curve button 78printing graphs 93

printing reports 99View Results screen, continued

Report button 77, 99report preview 99resizing panels 53Save Run button 43Select Graphs button 77Update Analysis button 78View Another Run button 44, 77Views lists 78

viewing cycle threshold results 89viewing melt peak results 89viewing multiple graphs 90viewing runs 77Views lists

analysis settings 78graph names 90protocols 90Results Table 85–90using 78

Wwarning messages 193–195Windows

archiving runs 220backing up SmartCycler database 220changing date or time 220default login 220desktop 220logging in as an administrator 220logging off 220performing database functions 220restoring SmartCycler database 220retrieving runs 220setting power schemes 220

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Index

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