Soft And Handling

Creating a Run

1. Click the Create Run icon

2. Enter a unique run name

3. Select dye set

4. Click the Add/Remove sites button

5. Select the protocol(s)-select a different protocol for each site.

6. Select sites –use ctrl and shift to select multiple sites

7. Click right-pointing arrow to transfer protocol and sites to the selections column.

8. Verify that selection is correct then click OK.

Defining a Protocol

2. Click New Protocol button

1. Click Define Protocols icon

3. Enter a unique protocol name and click OK.

4. Enter thermal cycling protocol.

5. Click Save Protocol

Advance to Next Stage feature – the protocol will automatically advance to the next PCR stage after the threshold crossing.

Defining a Graph

1. Click Define Graphs icon

2. Click New Graph button

3. Enter a unique graph name and click OK.

4. Enter graph definition

5. Click Save Graph

Viewing Results

Compare two runs

Customize Views list

View temperature and optical data in real time

Customize data analysis

Viewing Results Table

• Setup standard curves

• Sample IDs

• View Ct values

• View Melt Temperatures

Background subtraction

•The background is subtracted initially at cycle 13

•Background Min cycle is 5

•At least 5 cycles are required for this calculation

•To avoid using fluorescence data derived from amplified DNA, the 4 most recent cycles are not used

•The background subtraction calculation stops 4 cycles before the threshold crossing.


Background subtraction and drift correction started at cycle 5 and stopped at cycle 28.39.


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

Threshold set too highBackground subtraction

Smart Cycler Menus

Smart Cycler Menus• Administration

•Login/Logout

•Search by run name or specimen

•Limit access to runs, protocols and graphs

• Customization•Analysis Settings

•Export data (jpeg and excel files)

•Melt analysis

• Automatic backup of database

Melt Settings

Control

•Internal Control –Used to validate an assay confirming that the reagents are functional.

•Quantitative Internal Control–Used to correct for differences in assay performance due to normal site-to-site and run-to-run variations.

Quantification Strategy•Absolute –requires standard whose concentration is known absolutely

–uses standard curve–unnecessary for most studies

•Relative –requires reference gene–uses normalization–Assumption that control doesn’t vary under the experimental conditions.

Absolute Quantification

•The fact that this method relies on a set of knows is the reason it cannot be “absolute”. No matter what the source or how carefully it is measured, there is no way to know exactly how much or how many copies of a known template truly exists in a given well of a known sample.

Gene quantification using Real Time Quantitative PCR : an emerging technology hits the mainstream. David G. Ginzinger, Experimental Hematology 30 (2002) 503-512.

Relative Quantification

Current methods to determine exact numbers of molecules overcome the determination of the amplification rate by assuming identical amplification rates for a target DNA sequence and a standard of known quantity introduced into the experiment design, so that only the ratio of amplified products need be determined. Violations of the hypothesis of identical amplification rates for two sequences will result in a systematic bias in the experiment results that underestimates or overestimates the initial copy numbers.

Statistical Estimations of PCR Amplification Rates. Jean Peccoud and Christine Jacob.

Quantification

Unknown Sample

Threshold101

102

103

104

105

Quantification

(Roche FastStart DNA SYBR Green kit; each dilution run in triplicate)SYBR® Green – Standard Curve

Setting up a Standard Curve

• Select Sample Type• Enter standard concentration• Click Update Analysis button• View Standard Curve graph

Importing a Standard Curve



Imported standard curve is highlighted in yellow.

Saving an Imported Standard Curve

Quantitation of Unknowns

Maintenance Screen

Smart Cycler Menus

User Administration Login/Logout

Login

Smart Cycler Menus

Run and Specimen logs allow the user to search by run name or specimen name.

Run log

Run Report

Specimen report

Specimen report

Specimen report

User AdministrationUser Administration can be used

to:

•Sort runs by user name

•Identify protocols and graphs by

user name

•Limit access to your runs,

protocols and graphs

tamlyn

tamlyn

****

****

User Administration

Enter a User Name and Password

Configure UserSelect User name

User Rights.

Setup Menu- System Defaults• Customize default Analysis

Settings

• Automatic backup of

database

• Customize exported data

and set automatic export

• Customize melt analysis

• Limit user access to their

own runs, protocols and

graphs

General

Analysis Setting

Automatic Backup

Default Export Settings



Export graph data

Export graph data

Melt Settings

Melt Settings

Melt Settings

Melt Settings

Melt Settings

Melt Settings

Melt Settings

Access Option

Smart Cycler Menus

Smart Cycler Menus

•At Calibration:–Measure raw values with buffer

–Measure raw values with each pure dye.

–Determine ‘net’ dye signals for each pure dye and construct signal matrix.

–Invert matrix to determine ‘calibration’ matrix

•During a run–Measure raw data of unknown mix

–Subtract off stored ‘buffer’ values

–Multiply by calibration matrix to obtain ‘deconvolved’ calibrated signals

Optical Calibration Issues

Optical Calibration IssuesThe emission spectra of fluorescent dyes is quite broad.

A pure dye produces signal in more than one optical channel. For example, TET

produces raw signal in Ch1, Ch 2 and Ch 3:

Example of Raw Signals from Pure Dyes

0

500

1000

1500

2000

2500

3000

Ch1 FAM Ch2 TET Ch3 TAM Ch4 ROX

Optical Channel

Raw

Op

tica

l S

ign

al

FAM

TET

TAM

ROX

Optical Calibration Issues (cont)

After calibration, we want each pure dye to give a signal

of 1000 fluorescent units only in its appropriate channel:Example of Ideal Calibrated Signals from Pure Dyes

0

200

400

600

800

1000

1200


Optical Channel

Flu

ore

scen

t U

nit

s

FAM

TET

TAM

ROX

Optical Calibration Issues (cont)

In reality, we see some ‘dye crosstalk’ between the channels, so we might see data like below after calibration: Example of Typical 'Verification' Data

from Pure Dyes

0

200

400

600

800

1000

1200


Optical Channel

Flu

ore

scen

t U

nit

s

FAM

TET

TAM

ROX

Signal Analysis - Version 1 and 2

Typical ICORE Signal Matrix for FTTR25 Crosstalk RatioFAM TET TAMRA ROX FAM TET TAMRA ROX

Ch1 1339 426 -3 0 Ch1 0.32Ch2 315 1011 350 82 Ch2 0.31 0.35Ch3 21 523 1077 440 Ch3 0.49 0.41Ch4 2 7 780 2289 Ch4 0.34

Signal Matrix for FCTC25 - Version 2 Crosstalk RatioFAM CY3 TexRd CY5 FAM CY3 TexRd CY5

Ch1 3561 -5 -1 -2 Ch1 0.00Ch2 538 2017 124 6 Ch2 0.27 0.06Ch3 -1 88 2374 55 Ch3 0.04 0.02Ch4 2 -4 49 2220 Ch4 0.02

Signal Matrix for FTTC25 - Version 2 Crosstalk RatioFAM TET TexRd CY5 FAM TET TexRd CY5

Ch1 2075 252 5 4 Ch1 0.12Ch2 375 2386 95 5 Ch2 0.16 0.04Ch3 0 3 2398 59 Ch3 0.00 0.02Ch4 0 -2 48 2221 Ch4 0.02

Help Menus

Help Menus

Software Diagnostic

Software Diagnostic

c\smartcycler\tools\cyclerdiagnostics

Tools used by technicians/engineers to perform functional testing and trouble-shooting of the smartcycler

Software Diagnostic

Command Menu

Site(s) activation

table

Cycler Diagnostic

Rev.

Software DiagnosticGetDeviceSN :

Reports the serial number of the instrument, backplane, the CPU board, the instrument model number and bootcode revision number

Software Diagnostic

Software Diagnostic

Troubleshooting examples

Obtain the following required information:•Serial number of Computer•Serial number of Smart Cycler

Additional info (change form original)•Computer Model and OP•Version of Smart Cycler Software•Version of the Anti-Virus software

Troubleshooting Computer Problems

Problem description :• Was the software open when the problem occurred?• Was a run in progress when the problem occurred?• What applications were open when the problem occurred?• Is the computer networked?


General Computer Information:•SSQL •USB devices•Printers•Windows 2000

–Login –Set all power settings to Never

•Additional Software•The Smart Cycler software license allows the user to load the software on1 additional

computer for data analysis


Most Frequent :•Loss of communication:

–Using more than one block?–USB cables–Additional software/hardware –Networking/Internet –Norton Anti-virus –Power questions

•The computer is running slow •The Database Cannot be Connected •Windows 2000


Frequently Asked Questions

• Acceptable temperature range : 40C to 98C • Can I backup my data? Yes, individual runs can be archived and retrieved or the entire

database can be backed up

• What is the capacity of the database? 1.9GB

• Can I use VIC on the Smart Cycler? Smart Cycler II system must be recalibrated with the

user-defined Optical Calibration

• Can I fill the 100ul tube with 50ul? No

• Can I use 100ul and 25ul together in a single run? No

And more …

General Software Questions

Rules

1.White coat must be worn at all times in the laboratory. Coats are stored in the laboratory. Coats are removed before leaving the laboratory.

2.Never ingest any chemical from lab and, if a chemical is spilled on your hands, wash them immediately

3.You must ALWAYS thoroughly wash and scrub your hands before you leave the laboratory.

4.Follow the specific safety rules for each hazardous chemical. Each hazardous chemical will be given to you with specific instructions for its handling. These vary from chemical to chemical depending on the hazard each presents. Follow these instructions exactly and ask for assistance if you are unsure about how to proceed.

How remove gloves

1 2 3

Seize glove few centimeters from glove rims.

Remove glove till appearance.

With fingers wrapped in glove turned inside out take the second glove off…

4 5 6

…turn completly the second glove inside out and discard it

finish turn the first glove inside out and discard it

Wash your hands.

1 2 3

Roll up sleeves first and wet hands.

Collect one dose of liquid soap.

Rub hands together for 30 secondes, work all surfaces, insist on palms.

4 5 6

Thoroughly rinse.

Dry hands with disposable hand towel.

Use towel to turn off water and discard in open bin

Recommendations relating to hand washing

Soft And Handling

Technology