UltraClean™ Blood DNA Isolation Kit · 2017. 11. 22. · 1 UltraClean™ Blood DNA Isolation Kit (PROCESSES 1,000 ml OF BLOOD) INTRODUCTION Use this kit for isolating high quality

1

UltraClean™ Blood DNA Isolation Kit

(PROCESSES 1,000 ml OF BLOOD)

INTRODUCTION

Use this kit for isolating high quality genomic, mitochondrial, or viral DNA from 300 µl,3 ml or 10 ml of whole blood. Scale up the protocol to process any volume of blood up to1,000 ml. DNA is ready for restriction digest, amplification, hybridization, sequencing,and cloning applications*.

The UltraClean™ Blood DNA Isolation Kit is designed to isolate high molecular weightgenomic DNA from whole blood, buffy coat, bone marrow, cultured cells, buccal swabs and other body fluids. This kit is solution based and does not use spin filters. As a result,it is possible to isolate high molecular weight DNA, greater than 100 kb.

SUMMARY

The yield and quality of DNA can vary greatly depending on the individuals or other sources(bone marrow, non-human) and storage of blood. Reduced yields and poor quality may occur ifsamples are collected, stored or handled improperly.

PROCEDURE

Mammalian blood is composed of both non-nucleated red blood cells (RBC) and nucleated white blood cells (WBC). Only the WBCs contain DNA. To isolate cleangenomic DNA, the RBCs are first preferentially lysed in a hypotonic buffer. The WBCs are pelleted to separate them from the debris. Then they are extracted in a bufferwith anionic detergent that stabilizes the DNA by preventing DNase activity. RNase is used to remove RNA and protein is removed with a precipitation step. The free genomic DNA is recovered using isopropanol precipitation, washed with 70% ethanol toremove excess salt, and resuspended in TE buffer to ensure long term stability at -80oC.Because the procedure is so gentle, the DNA recovered will be of high molecular weight.

INSTRUCTION MANUAL

NO

TE This UltraClean™ Blood DNA Isolation Kit Instruction Manual includes specific

protocols that can also be used for isolating genomic DNA from a variety ofsample sources such as buffy coat, bone marrow, buccal cells and cultured cells.

*This kit is for research purposes only. Not for diagnostic use.

Version:06242008

Catalog No Description12000-1000 UltraClean™ Blood DNA Isolation Kit12002-1000 UltraClean™ Blood DNA Isolation Kit Plus RNase

2

KIT STORAGE AND STABILITY

Store RNase A Solution at 4°C (included in catalog# 12002-1000 only). All other kitreagents and components should be stored at room temperature (15-30°C). When storedas directed, this kit is stable until the date of expiration printed on label.

KIT CONTENTS

EQUIPMENT REQUIRED

Centrifuge (2,500 x g)Centrifuge (13,000 – 16,000 x g)Pipets & pipet tips (15 µl – 30 ml)Vortex Genie2Water Bath (65°C)2.0 ml microcentrifuge tubes; MO BIO catalog#1200-100-T (bag of 100)15 ml Collection Tubes; MO BIO catalog#12700-T (bag of 25)50 ml Centrifuge Tubes; MO BIO catalog#12600-T (bag of 25)Centrifuge tubes appropriate for the volume of blood processed. See protocol.

USER PROVIDED SOLUTIONS REQUIRED

100% Isopropanol (2-propanol); MO BIO catalog#12000-100-5 (33 ml) 70% Ethanol; MO BIO catalog#12000-100-6 (30 ml)RNase A Solution (25 mg/ml); MO BIO catalog#1202-1 (1 ml) and #1202-5 (5 ml) Proteinase K Solution (20 mg/ml); For use with bone marrow protocol only. MO BIOcatalog#1222-2 (2 ml)

NO

TE The UltraClean™ Blood DNA Isolation Kit Plus RNase (catalog# 12002-1000)

includes RNase A Solution. The UltraClean™ Blood DNA Isolation Kit (catalog# 12000-1000) does not include RNase A Solution.

Catalog No. Description Amount12000-1000-1 Solution G1 (RBC Lysis Solution) 3030 ml12000-1000-2 Solution G2 (Cell Lysis Solution) 1010 ml12000-1000-3 Solution G3 (Protein Precipitation Solution) 410 ml12000-1000-4 Solution G4 (DNA Hydration Solution) 70 ml

Catalog No. Description Price

12002-1000-1 Solution G1 (RBC Lysis Solution) 3030 ml12002-1000-2 Solution G2 (Cell Lysis Solution) 1010 ml12002-1000-3 Solution G3 (Protein Precipitation Solution) 410 ml12002-1000-4 Solution G4 (DNA Hydration Solution) 70 ml12002-1000-5 RNase A Solution (25 mg/ml) 5 ml


UltraClean™ Blood DNA Isolation Kit Plus RNase

3

WARNINGS AND PRECAUTIONS

1. This kit is intended For Laboratory Use Only.

2. Avoid all skin contact with reagents in this kit. In case of contact, wash thoroughlywith water. Do not ingest. See Material Safety Data Sheets for emergency procedures in case of accidental ingestion or contact.

3. The Center for Disease Control, the Food and Drug Administration, and theAmerican Hospital Association recommend “universal precautions” when workingwith blood and body fluids. To prevent contact with potentially infectiouspathogens, it is recommended that workers protect themselves from contact withthese fluids by using suitable barrier protection which includes gloves. Effectivebarrier protection should be used during this procedure.

4. All MSDS information for all UltraCleanTM Blood DNA Isolation Kits andcomponents is available upon request (760-929-9911) or at www.mobio.com.

PRODUCT WARRANTY AND RETURN POLICY

MO BIO guarantees the performance of all product in the manner described in ourproduct literature. The Purchaser must determine the suitability of the product for itsparticular use. Should any product fail to perform satisfactorily due to any reason otherthan misuse or improper storage, MO BIO will replace it free of charge or refund thefull purchase price of the product. Requests for return authorization must be madewithin 20 days after receipt of the products or if the product was damaged during shipping, within 3 days after receipt of the product. Products ordered in error may bereturned within 10 days after receipt of the product. MO BIO cannot accept returnswithout prior authorization.

Shipping costs will not be credited and return shipping costs will be paid by the customer. A 20% restocking fee will be applied. Please contact our Customer ServiceDepartment at 1-800-606-6246 (US) or 1-760-929-9911 (outside US) and provide uswith the purchase order number, our reference or invoice number, catalog number anddescription of the product, and reason for return.

ULTRACLEAN™ BLOOD DNA ISOLATION KIT QUALITY ASSURANCE

MO BIO Laboratories, Inc. strives for the highest level of quality for you the customer. Our documented and very rigorous manufacturing procedures are equal toor exceed most ISO and GMP standards. We will provide you with certificates of analysis for any batch lot we manufacture upon request. We documented end-usetesting of all reagents, and components used in our kits or other products.

EXPECTED RESULTS

The UltraCleanTM Blood DNA Isolation Kit is designed to meet performance specificationsfor DNA yield and quality.

• Expected yield will range between 25-40 µg of DNA/ml of healthy human blood.

• DNA molecular weight is up to 200 kb with minimal shearing.

• A260/A280 – 1.8-2.0

• DNA is ready for PCR, restriction digests, and all other downstream applications.

The UltraClean™ Blood DNA Isolation Kit is designed to purify high quality DNA inhigh yield.The table below shows the expected DNA yield range from a variety of samplesources.The actual yield obtained will depend on the sample genome size, sample type andnumber of cells in the sample.

4

Sample Type Yield Range Yield AverageWhole Blood 16-50 µg/ml blood 35 µg/ml bloodCulture Cells 5-10 µg/1-2 million cells 7 µg/1 million cellsBuccal Swabs 0.2–2 µg/swab 1 µg/swabBone Marrow 5-10 µg/1-2 million cells 7 µg/1 million cells

Expected DNA Yield Range from a Variety of Sample Types

5

HUMAN AND MAMMALIAN WHOLE BLOOD, BONE MARROWAND BUFFY COAT SAMPLE COLLECTION AND HANDLING

WHOLE BLOOD AND BONE MARROW

1. If possible, collect whole blood and bone marrow in EDTA tubes to reduceDNA degradation. However, other anticoagulants such as ACD (citrate) andheparin may also be used successfully.

2. Store fresh samples at 4°C for up to 5 days for highest yields. Blood will be stable for several months and provide sufficient DNA for most applications suchas PCR.

3. Frozen samples are stable at -80°C for at least two years. Before use, thaw quicklyin a 37°C water bath and keep sample on ice until use.

BUFFY COAT

For DNA extraction, EDTA tubes are preferred to prevent DNA degradation. However,other anti-coagulants, such as ACD or heparin may also be used. Store fresh samples at 4°C.

Centrifugation Method:

1. To obtain the WBC layer, centrifuge whole blood at 1,500-2,000 x g (with thebreak off) for 10-15 minutes at room temperature. The blood will separate intoplasma at the top and red blood cells at the bottom with a white interfacebetween them called the buffy coat.

2. Remove the upper plasma layer with a Pasteur pipette to within 1 mm of the WBC layer. Discard the plasma if you are not using it for other studies.

3. Using the Pasteur pipette, carefully suction off the white layer in as small a volume as possible. Contamination with some of the RBCs is expected.

4. Count the cells using a hemacytometer.Wash with PBS and distribute into singleuse aliquots and centrifuge to pellet. Cells may be frozen or used immediately for extraction.

Ficoll-Hypaque Method (Fisher Scientific catalog# NC 9524199):

1. Add 3 ml of Ficoll to a 10 ml round bottom or 15 ml Falcon tube.

2. Layer 3 ml of whole blood on top of the Ficoll layer by pouring carefully downthe side of the tube. Do not mix the Ficoll with blood.

3. Centrifuge at 2,000 x g (with the break off) for 15 minutes at 4°C or room temperature.

4. The buffy coat will be at the interphase between the upper plasma layer and the lower RBC layer. Use a Pasteur pipette to remove the interphase layer containing the WBCs.

5. Wash the cells in PBS twice and count using a hemacytometer. Aliquot for single use before freezing.

6

HELPFUL HINTS FOR WORKING WITH LARGE NUMBERS OF SAMPLES

The following are hints for high-throughput sample processing using the UltraClean™ Blood DNA Isolation Kit. Please contact MO BIO Technical Services with any questions or for additional information.

1. Use multi-channel pipettors to dispense Solution G1 (RBC Lysis Solution),Solution G2 (Cell Lysis Solution), Solution G3 (Protein PrecipitationSolution), 100% Isopropanol, 70% Ethanol and Solution G4 (DNA Hydration Solution). Using Repipettors helps prevent contamination ofreagents and saves time.

2. Pre-aliquot Solution G1 (RBC Lysis Solution) and 100% Isopropanol.

3. Lyse the cells in Solution G2 (Cell Lysis Solution) using a vortex instead of pipetting.

4. Use a multi-tube vortex to vortex 24 samples at once (vortex adapter for Genie 2(220 V: cat # 13111-V-220).

5. Add RNase A Solution to Solution G2. RNase A is stable in Solution G2 for atleast 8 weeks at room temperature.

6. Precipitate the multiple samples with 100% Isopropanol simultaneously byinverting all samples in the rack.

7. After rehydration, transfer samples purified in 15 ml or 50 ml centrifuge tubesto 2 ml microcentrifuge tubes.

8. A rotator may be used to gently mix samples during incubations in Solution G1,100% Isopropanol, and Solution G4.

7

WHOLE BLOOD AND BUFFY COAT REAGENT VOLUMES SCALING TABLE:

(0.05 ml to 10 ml Blood or 0.35 to 70 million White Blood Cells)

À These are the number of expected white cells from the original volume of blood.Cell number estimates assume an average of seven million white cells per ml ofwhole blood. For buffy coat samples, expect this number of cells from the volume of blood at the top of this column.

Á For whole blood use 3 volumes of Solution G1 (RBC Lysis Solution) for every volume of blood. For buffy coat, use 3 volumes of Solution G1 for every 1 volume of buffy coat. For example, 3 ml of Solution G1 should be used to lysered blood cells in 1 ml of buffy coat.

Â If the cell count is expected to be

Number of CellsÀ 100-10,000 0.5-1 million 3-5 million 30-50 millionTube Size 0.6 ml 2 ml 2 ml 15 mlSolution G2 (ml) 0.06 0.15 0.6 6RNase A Solution (µl) 0.5 0.75 3 30

Solution G3 (ml) 0.02 0.033 0.2 2100% Isopropanol (ml)Á 0.06 0.15 0.6 6

70% Ethanol (ml) 0.06 0.15 0.6 6Solution G4 (µl)Â 10 25 100 500Expected DNA Yield Range (µl)Ã 0.0004-0.04 2.0-6.0 15-30 80-300

8

CULTURED CELLS REAGENT VOLUMES SCALING TABLE:

(100 Cells to 100 million Cells)

À Count the cells using a hemacytometer or other cell counter.

Á If the cell count is expected to be ≤20,000 cells, add a DNA carrier such asglycogen (1µl Glycogen Solution (20 mg/ml) per 600 µl Isopropanol).

Â Solution G4 (DNA Hydration Solution) volume is a suggested volume. SolutionG4 may be adjusted according to the desired concentration and based on expectedDNA yield.

Ã DNA Yield is based on an estimate of 6 pg DNA per human diploid cell and 80% recovery.

VIRAL DNA PURIFICATION

Virus must be white blood cell-associated for the whole blood or buffy coat protocol toefficiently isolate DNA.

9

DNA PURIFICATION RESULTS


(PROCESSES 1,000 ml OF BLOOD)

Figure 1: DNA was successfully isolated from avariety of samples with the UltraClean™ Blood DNA Isolation Kit. Lane 1: Marker; Lane 2: fresh whole blood; Lane 3: frozen whole blood; Lane 4: buccal swab;Lane 5: bone marrow; Lane 6: culture cells

Figure 2: Restriction Enzyme Digestion of DNA isolated with UltraClean™ Blood DNA Isolation Kit.Lane 1: Marker; Lane 2: Eco RI; Lane 3: Hae III;Lane 4: Hind III

Figure 3: PCR using the following primer sets onDNA isolated from different volumes of blood usingthe MO BIO UltraClean™ Blood DNA Isolation Kit:Primer: Amel-A (Lane 1 and Lane 2) – malePrimer: Amel-B (Lane 3 and Lane 4) – female

(X-Y homologous amelogenin gene product for femalesex determination, 106 bp for X chromosome, andproduct 112 bp for Y chromosome.)

10

Whole BloodProcedure

Buccal SwabProcedure

Bone MarrowProcedure

Air dryResuspend DNA inDNA Hydration Solution

TOTAL GENOMIC DNA

SelectiveRBC Lysis

SelectiveRBC LysisBuccal swab

Protein Precipitation

Add isoprapanol

Wash 1x

DNA Pellet

DNA Hydration

DNA Precipitation

LeukocytePellet

Bone MarrowCell Pellet

Cell Lysis + RNase Atreatment (optional)

11

PROTOCOL – DNA PURIFICATION FROM 300 µl OF BLOOD

Expected yields from 300 µl of blood is 5 – 15 µg of DNA.This prep may be scaled up to 15 ml – 50 ml tubes if a larger starting volume of blood is used. Scale up solution volumesaccordingly. For 3 ml and 10 ml preps, see the following pages for the appropriate protocol.

Please wear gloves at all times.

SAMPLE PREPARATION

See Sample Collection and Handling section (page 5).

CELL LYSIS

For frozen samples, thaw quickly at 37°C and place immediately on ice. Blood stored at4°C for several months may be used with good results.

1. Add 300 µl of whole blood to 900 µl of Solution G1 (RBC Lysis Solution).

2. Invert twice and incubate 5 minutes at room temperature. Invert two additionaltimes during incubation.

3. Centrifuge for 30 seconds at 13,000 x g. Remove supernatant with a narrowpipet tip and discard without disturbing the pellet. (About 20 µl of supernatant will remain.)

4. Bump vortex to resuspend the pellet completely.

5. Check Solution G2 (Cell Lysis Solution). If precipitated, heat to 55-65°C for 5 minutes to dissolve. Add 300 µl of Solution G2.

6. Pipet up and down to lyse the cells. If cell clumps are still visible incubate at 37°Cuntil no clumps are visible. Caution: excessive pipetting at this step can lead tosheared genomic DNA.

NOTE: Samples will be stable at room temperature in Solution G2 for extended periods; up to 8 months. Sample must be at room temperature before proceeding.

RNase TREATMENT (OPTIONAL)

7. Add 1.5 µl of RNase A (25 mg/ml), invert 5 times and vortex on low speed for 5 seconds. No incubation is required.

NOTE: If using MO BIO catalog# 12000-1000 see User Provided SolutionsRequired section for RNase A ordering information on page 2.

PROTEIN PRECIPITATION

8. Add 100 µl of Solution G3 (Protein Precipitation Solution).

9. Immediately vortex on high speed setting for 15 seconds.

10. Centrifuge 3 minutes at 13,000 x g. If you do not see a pellet, refer to theTroubleshooting Guide.

12

DNA PRECIPITATION

11. Remove clear supernatant and transfer to a clean 2.0 ml microcentrifuge tube.

12. Add 300 µl of 100% Isopropanol.

13. Invert 15 times and incubate at room temperature for 3 minutes.

14. Centrifuge 1 minute at 13,000 x g.

15. Carefully pour off supernatant without disturbing pellet and drain on a papertowel or absorbent material.

16. Add 300 µl of 70% Ethanol.

17. Invert tube 5 times to wash.

18. Centrifuge 30 seconds at 13,000 x g.

19. Carefully pour off supernatant without disturbing the pellet and drain on a papertowel or absorbent material.

20. Allow to air dry until the ethanol is completely evaporated (about 5-10 minutes).

DNA HYDRATION

21. Add 100 µl of Solution G4 (DNA Hydration Solution).

22. Heat in 65°C water bath tapping tube occasionally until pellet has been completely resuspended (should take no longer than 30-40 minutes).

23. Centrifuge sample for 5 seconds at 10,000 x g at room temperature to gather allthe liquid in the bottom of the tube.

24. Genomic DNA in the tube is now ready to use for any application.NOTE: Store DNA at 4°C. For long term storage, store at -20°C or -80°C.

Thank you for choosing the UltraClean™ Blood DNA Isolation Kit.

13

PROTOCOL – DNA PURIFICATION FROM 3 ml OF BLOOD

Expected yields from 3 ml of blood is 50 – 150 µg of DNA.

This prep may be scaled up or down accordingly.


SAMPLE PREPARATION


CELL LYSIS


1. Add 3 ml of whole blood to 9 ml of Solution G1 (RBC Lysis Solution) in a 15 ml centrifuge tube.


3. Centrifuge for 5 minutes at 2,500 x g. Remove supernatant with a narrow pipettip and discard without disturbing the pellet. (About 200 µl of supernatant will remain.)


5. Check Solution G2 (Cell Lysis Solution). If precipitated, heat to 55-65°C for 5 minutes to dissolve. Add 3 ml of Solution G2.

6. Pipet up and down to lyse the cells. If cell clumps are still visible, incubate at37°C until no clumps are visible. Caution: excessive pipetting at this step can lead to sheared genomic DNA.

NOTE: Samples will be stable at room temperature in Solution G2 for extendedperiods; up to 8 months. Sample must be at room temperature before proceeding.


7. Add 15 µl of RNase A (25 mg/ml), invert 5 times and vortex on low speed for 5 seconds. No incubation is required.

NOTE: If using MO BIO catalog# 12000-1000 see User Provided Solutions Required section for RNase A ordering information on page 2.


8. Add 1 ml of Solution G3 (Protein Precipitation Solution).


10. Centrifuge 5 minutes at 2,500 x g. If you do not see a pellet, refer to theTroubleshooting Guide.

14

DNA PRECIPITATION

11. Remove clear supernatant and transfer to a clean 15 ml centrifuge tube.

12. Add 3 ml of 100% Isopropanol.


14. Centrifuge for 10 minutes at 2,500 x g.

15. Carefully pour off supernatant without disturbing pellet and drain on a papertowel or absorbent material.

16. Add 3 ml of 70% Ethanol.



19. Carefully pour off supernatant without disturbing the pellet and drain on a papertowel or absorbent material.


DNA HYDRATION


22. Incubate at 65°C for 1 hour. DNA samples can be incubated at room temperature overnight. Tap the tube at least twice during the incubation.

23. Centrifuge the tube containing DNA sample for 20 seconds and transfer to a microcentrifuge tube.

24. Genomic DNA in the tube is now ready to use for any application.

NOTE: Store DNA at 4°C. For long term storage, store at -20°C or -80°C.


15

PROTOCOL – DNA PURIFICATION FROM 10 ml OF BLOOD

Expected yields from 10 ml of blood is 150 – 500 µg of DNA.

This prep may be scaled up or down accordingly.


SAMPLE PREPARATION


CELL LYSIS


1. Add 10 ml of whole blood to 30 ml of Solution G1 (RBC Lysis Solution) in a50 ml centrifuge tube.


3. Centrifuge for 5 minutes at 2,500 x g. Decant by pouring off the supernatant anddiscard without disturbing the pellet. (About 200 µl of supernatant will remain.)

4. Bump vortex to resuspend the cell pellet completely in the residual liquid.

5. Check Solution G2 (Cell Lysis Solution). If precipitated, heat to 55-65°C for 5minutes to dissolve. Add 10 ml of Solution G2.

6. Pipet up and down to lyse the cells. If cell clumps are still visible, incubate at37°C until no clumps are visible. Caution: excessive pipetting at this step canlead to sheared genomic DNA.



7. Add 45 µl of RNase A (25 mg/ml), invert 5 times and vortex on low speed for 5 seconds. No incubation is required.

NOTE: If using MO BIO catalog# 12000-1000 see User Provided SolutionsRequired section for RNase A ordering information on page 2.


8. Add 4 ml of Solution G3 (Protein Precipitation Solution).


10. Centrifuge for 5 minutes at 2,500 x g. If you do not see a pellet, refer to theTroubleshooting Guide.

16

DNA PRECIPITATION

11. Remove clear supernatant and transfer to a clean 50 ml centrifuge tube.

12. Add 10 ml of 100% Isopropanol.



15. Carefully pour off supernatant without disturbing pellet, and drain on a papertowel or absorbent material.

16. Add 10 ml of 70% Ethanol.



19. Carefully pour off supernatant without disturbing the pellet and drain on apaper towel or absorbent material.


DNA HYDRATION


22. Incubate at 65°C for 1 hour. DNA samples can be incubated at room temperatureovernight. Tap the tube at least twice during the incubation.

23. Centrifuge the tube containing DNA sample for 20 seconds and transfer to amicrocentrifuge tube.




17

PROTOCOL – DNA PURIFICATION FROM BUFFY COAT

SAMPLE PREPARATION


1. The buffy coat can be collected from 3 ml of whole blood by direct centrifugation.NOTE: If the buffy coat contains any red blood cells (RBC) continue with the

RBC Lysis procedure (step 2). If the buffy coat sample is free of RBCs, aliquot 200-300 µl of sample into a 15 ml centrifuge tube and skip to the Cell Lysis procedure (step 5).

RBC LYSIS

2. To 200-300 µl of buffy coat sample in a 15 ml conical centrifuge tube, add 3 volumes of Solution G1 (RBC Lysis Solution). Mix and incubate at room temperature for 5 minutes. Invert one additional time during incubation.

3. Centrifuge at 2,500 x g for 5 minutes. Remove supernatant with a narrow pipettip without disturbing the pellet. (About 100-150 µl of supernatant will remain.)


CELL LYSIS

5. Check Solution G2 (Cell Lysis Solution). If precipitated, heat to 55-65°C.Add 3 ml of Solution G2 to the buffy coat.

6. Pipette up and down to lyse the cells. If cell clumps are still visible, incubate at37°C until no clumps are visible. Caution: excessive pipetting at this step canlead to sheared genomic DNA.



7. Add 3 µl of RNase A (25 mg/ml) to the cell lysate.


8. Mix sample by inverting several times. Incubate the tubes for 15 minutes at 37°C.


9. Incubate sample on ice for 1 minute to quickly cool the sample.

10. Add 1 ml of Solution G3 (Protein Precipitation Solution) and vortex at highspeed for 20 seconds.

11. Incubate on ice for 5 minutes. Centrifuge at 2,500 x g for 5 minutes.

18

DNA PRECIPITATION

12. Collect the supernatant containing DNA and transfer to a clean 15 ml centrifuge tube. Add 3 ml of 100% Isopropanol.

13. Mix by gently inverting two times. Incubate at room temperature for at least 3 minutes.

14. Centrifuge at 2,500 x g for 10 minutes.

15. Carefully discard the supernatant and drain the tube by inverting on a cleanpiece of absorbent paper.

16. Add 3 ml of 70% Ethanol and invert 5-10 times to wash the DNA pellet.

17. Centrifuge for 5 minutes at 2,500 x g. Carefully decant the ethanol. Drain the tube by inverting on a piece of clean absorbent paper. Air dry for 15 minutes.

DNA HYDRATION



20. Centrifuge the tube containing the DNA sample for 20 seconds and transfer to amicrocentrifuge tube.




19

PROTOCOL – DNA PURIFICATION FROM BONE MARROW CELLS

NOTE: DNA purification from bone marrow cells requires the use of Proteinase K Solution (20 mg/ml); MO BIO catalog# 1222-2.

SAMPLE PREPARATION


BONE MARROW CELL COLLECTION

Wear eye protection. Use this procedure to flush marrow cells from small sections ofmouse bones. The following protocol is optimized for 4 – 8 mouse femur bone shafts. Ifyou already have a bone marrow cell pellet, start with the Cell Lysis step. If you have marrow cells in suspension, pellet cells (steps 5 and 6 below).NOTE: You will need a sterile solution of phosphate buffered saline (PBS). Also useful is

a cell strainer: BD Falcon catalog# 352350.

1. Scrape away tissue with sterile scalpel until bone is free of any tissue. Using sterile scissors, cut off both rounded ends of the bone. Cut as close to the socketas possible. Avoid shattering the shaft.

2. Place bone in clean Petri dish containing PBS to keep bone moist until ready to process.

3. Using sterile forceps or a hemostat, remove the bone shaft and hold over a clean60 mm Petri dish. Using a 3 cc syringe and needle (25 gauge) filled with sterilePBS push the needle into the marrow and flush marrow cells out through theopposite end of the bone shaft. Bone should lose its pinkish red color andbecome white or translucent when the marrow has been completely removed.

4. Pipet the solution containing cells into a 50 ml conical tube (for optimal resultsstrain cell material through a cell strainer to remove any large particles). To maximize the number of cells collected, be sure to rinse the cell strainer and Petri dish with extra PBS.

5. Centrifuge at low speed at 1,400 rpm (400 x g) for 5 minutes at room temperature.

6. Carefully discard the supernatant without disturbing the pellet.

7. Centrifuge again at 400 x g for 1 minute and carefully remove all traces of remaining supernatant.

CELL LYSIS

8. Resuspend the cell pellet with 300 µl of Solution G1 (RBC Lysis Solution) andtransfer to a clean 2.0 ml microcentrifuge tube.

9. Incubate for 1 minute at room temperature, invert gently two times during this incubation.

10. Centrifuge for 30 seconds at 13,000 x g. Remove 280 µl of supernatant carefullyusing a pipet tip leaving behind 20 µl of supernatant.


20

12. Check Solution G2 (Cell Lysis Solution). If precipitated, heat to 55-65°Cfor 5 minutes to dissolve. Add 300 µl of Solution G2.

13. Pipet up and down to lyse the cells. Caution: excessive pipetting at this step canlead to sheared genomic DNA.


14. Add 1.5 µl of Proteinase K Solution (20mg/ml) and incubate at 55°C for 15-30minutes with inversion at least once during the incubation.


15. Add 1.5 µl of RNase A (25 mg/ml) to the cell lysate.

NOTE: If using MO BIO catalog# 12000-1000 see User Provided SolutionsRequired section for RNase A ordering information.

16. Mix the sample by inverting the tube 5 times and incubate at 37°C for 15-30 minutes.


17. Add 100 µl of Solution G3 (Protein Precipitation Solution) and vortex to mix.

18. Centrifuge at 13,000-16,000 x g for 5 minutes.

DNA PRECIPITATION

19. Remove clear supernatant and transfer to a clean 2 ml microcentrifuge tube.

20. Add 300 µl of 100% Isopropanol, mix the sample by inverting 15 times and incubate at room temperature for 3 minutes.

21. Centrifuge at 13,000 x g for 5 minutes and carefully discard the supernatant.A small DNA pellet should be visible.

22. Add 300 µl of 70% Ethanol.

23. Invert tubes 5 times to wash the pellet.

24. Centrifuge at 13,000 x g for 1 minute.

25. Invert and drain the tube on a clean absorbent paper and air dry for 1-2 minutes.

DNA HYDRATION



28. Centrifuge the tube containing DNA sample for 20 seconds.

29. Genomic DNA in tube is now ready to use for any application.NOTE: Store DNA at 4°C. For long term storage, store at -20°C or -80°C.


21

PROTOCOL – DNA PURIFICATION FROM BUCCAL CELLS

SAMPLE PREPARATION

1. To collect buccal cells, scrape the inside of the mouth 10 times with a sterile buccal cell collection brush or a cotton swab. For best results, wait at least 1 hourafter eating or drinking to collect buccal cells.

2. If DNA is not isolated immediately, samples may be stored on the collectionbrush or swab for up to two weeks at room temperature.

CELL LYSIS

3. Check Solution G2 (Cell Lysis Solution). If precipitated, heat to 55-65°C for 5 minutes to dissolve. Dispense 300 µl of Solution G2 into a 2.0 ml microcentrifuge tube.

4. Remove the collection brush head using sterile scissors or razor blade and placethe detached head in the tube. Alternately, if you are using a swab, insert the swabinto Solution G2 and mix gently up and down to dislodge the cells in to the solution.

5. Incubate the sample at 65°C for at least 15 minutes. If maximum yield isrequired, add 1.5 µl of Proteinase K Solution (20 mg/ml) (MO BIO catalog # 1222-2), mix by inverting two additional times and incubate at 55°C for atleast 1 hour.


6. Remove the collection brush head from the Solution G2, scraping and pressingit on the insides of the tube to squeeze out and recover as much liquid as possible.


7. Add 1.5 µl of RNase A (25 mg/ml) to the cell lysate.


8. Mix sample by inverting several times. Incubate the tubes for 15 minutes at 37°C.


9. Incubate the sample on ice for 1 minute to quickly cool.

10. Add 100 µl of Solution G3 (Protein Precipitation Solution) to the cell lysate and vortex vigorously for 20 seconds.

11. Incubate on ice for 5 minutes. Centrifuge for 3 minutes at 13,000 -16,000 x g.The precipitated proteins should form a tight pellet. If the pellet is not tight,incubate again on ice for 5 minutes and repeat the centrifugation.

22

DNA PRECIPITATION

12. Collect the supernatant containing DNA and transfer to a clean 2.0 ml microcentrifuge tube. Add 300 µl of 100% Isopropanol. If the DNA yield is expected to be low (

23

PROTOCOL – DNA PURIFICATION FROM 1-2 MILLIONCULTURED CELLS

SAMPLE PREPARATION


1. Cultured cells can be collected in suspension and processed immediately or kepton ice until ready to process. Alternatively cell pellets can be stored frozen.

2. For freezing the cells, perform steps 5 and 6 of the Cell Lysis procedure below.Freeze the pellets at -70°C. Cell pellets are stable at -70°C to -80°C for at least a year.

3. For purifying DNA from frozen samples, thaw samples and keep on ice. Proceedwith step 7 of the Cell Lysis procedure below.

4. Count the number of cells using a hemacytometer.

CELL LYSIS

5. Transfer 1-2 million cells in culture medium to a 1.5 ml microcentrifuge tube.The volume processed will vary based on the cell count.

6. Centrifuge at 12,000-14,000 x g for 10 seconds to pellet cells. Decant supernatant using a pipette tip leaving behind 10-20 µl residual liquid.

7. Resuspend the cells by vortexing the tube on high speed.

8. Check Solution G2 (Cell Lysis Solution). If precipitated, heat to 55-65°C for 5 minutes to dissolve. Add 300 µl of Solution G2 to the resuspended cells andvortex on high speed for 10-15 seconds to lyse the cells. If cells are not lysedcompletely after vortexing or if cell clumps are seen in the sample, incubate at37°C until clear lysate is visible.



9. To the cell lysate, add 1.5 µl of RNase A (25 mg/ml).


10. Invert the sample several times and incubate at 37°C for 15-20 minutes.Incubation can be extended up to 60 minutes if desired.


11. Place the sample on ice for 1 minute to cool to room temperature.

12. Add 100 µl of Solution G3 (Protein Precipitation Solution) to the RNase A-treated sample.

13. Vortex at high speed for 25 seconds to uniformly mix the sample.

14. Centrifuge at 13,000-16,000 x g for 2 minutes.The precipitated proteins shouldform a tight pellet. If the precipitated proteins do not form a tight pellet, vortexthe sample vigorously at high speed for 25 seconds again and incubate on ice for5 minutes, and repeat the centrifugation step.

DNA PRECIPITATION

15. Collect the supernatant from step 14 in a clean 2.0 ml microcentrifuge tube.Add 300 µl of 100% Isopropanol. Invert the sample tubes gently several times.

16. Centrifuge at 13,000 x g for 2 minutes. A small white pellet should be visible.

17. Decant the supernatant and invert the tube on clean absorbent paper to remove excess isopropanol. Add 350 µl of 70% Ethanol and invert the tube several timesto wash the DNA pellet.

18. Centrifuge at 13,000 x g for 2 minutes. Decant the Ethanol slowly to avoid losing the pellet.

19. Drain the tube on a clean absorbent paper for 10-15 minutes.

DNA HYDRATION

20. Add 50 µl of Solution G4 (DNA Hydration Solution), vortex for 5 seconds at medium speed to mix.

21. Incubate at 65°C for 1 hour. DNA Samples can also be incubated at room temperature overnight with gentle shaking to aid in hydration.

22. Genomic DNA in tube is now ready to use for any application.NOTE: Store DNA at 4°C. For long term storage, store at -20°C or -80°C.


24

25

FREQUENTLY ASKED QUESTIONS (FAQ’S) FOR ULTRACLEAN™ BLOOD DNA ISOLATION KIT USERS

PROTOCOL QUESTIONS

1. My starting material is different than the sample types mentioned in the MO BIO protocols. Can I still use the MO BIO reagents from the kit?

Yes. Some tough to lyse samples such as mucous body secretions, semensamples, etc. will require some pretreatments. Please check with our TechnicalServices Department for more information.

2. Should I use Glycogen? If so, when should I use it?

Glycogen should be used during the DNA precipitation step if expected yield isless than 2 µg as recommended in the Buccal Cell Protocol. Use with samplesthat may result in a low yield of DNA (for example low numbers of buccal cells,compromised samples and body fluids).

3. My centrifuge settings are in RPM, how can I convert it in to g-force?

All centrifuge steps are described in g-force. RPM and g-force are two differentunits. Make sure samples are centrifuged at the correct g-force. Failure to do somay result in lower DNA yields, protein contamination or even loss of pellets.Use the equation below to set the centrifuge to ensure the exact spinning speed:g-force= 1.12 x r x (rpm/1000)2.NOTE: r = radius of rotor in millimeters from center of rotor to center of tube cavity.

4. There is still some liquid in the tubes after the 10 minute drying period.Should I let them dry for a longer time?

If drying the DNA samples for 10 minutes is not sufficient, drying can beextended for a slightly longer period without affecting the hydration (1-2 hours).Avoid overnight drying; this might result in difficulty in resuspending DNA inthe hydration buffer.

KIT QUESTIONS

1. Where can I buy the alcohol solutions: 70% Ethanol and 100% Isopropanol?

Both of the alcohol solutions may be purchased by either directly contacting MO BIO or your local MO BIO distributor. Please see below for the catalognumbers: Cat # 12000-100-6 (70% Ethanol, 30 ml bottle), Cat # 12000-100-5(100% Isopropanol, 33 ml bottle).

2. What are the ingredients and formulation of the reagents?

All the formulations of the reagents in the UltraClean™ Blood DNAIsolation Kit are proprietary except for Solution G4 (DNA Hydration Solution) which is 10 mM Tris, 1 mM EDTA pH 8.0. Please refer to the MSDS for safety information.

26

3. My reagents were frozen when I received them. Can I still use them?

Yes. Once you thaw the reagents and mix them well, you can use them as normal.If any reagent has formed a precipitate, heat to 65°C until clear.

4. The Glycogen and/or the RNase A Solution were left out at room temperature. Can I still use them?

Yes. However, make sure to place these items at their correct storage temperature.

Please see below for more information:a. RNase A – Stability and storage: The enzyme is stable for years stored as a

refrigerated dry powder or frozen in solution; refrigerated solutions are stablefor weeks.NOTE: RNase A aggregates upon lyophilization and storage.

b. PROTEINASE K – Stability and storage: Lyophilized: 1-2 yearsAqueous stock solutions: 72 hours at +4°C or 4 months as a frozen solution.Concentrated stock solutions: 1-2 years at -20°C.

c. GLYCOGEN – Storage: Solution should be aliquoted into suitable volumes and stored at -20°C.

5. There is a white flaky precipitate, in the Cell Lysis Solution. Can I still use this solution?

Yes. The SDS present in Solution G2 (Cell Lysis Solution) will precipitate atcooler temperatures. Warm the Cell Lysis Solution in a water bath at 55-65°Cuntil the precipitate re-dissolves. Mix well before use.

6. Can I get a Material Safety Data Sheet (MSDS)?

The MSDS sheets are available on our website. Each product page has a corresponding MSDS. You can also request them through Technical Services.These can be sent via fax, email or mail.

7. Can I get a Certificate of Analysis (C of A) for the UltraClean™ Blood DNA kit?

Yes.You can request a C of A from MO BIO’s Technical Services. Please makesure to provide the catalog number and lot number of the solution and/or kit C of A being requested. This C of A can be faxed, e-mailed or mailed to you.

27

TROUBLESHOOTING GUIDE

RBC LYSIS STEP

1. The lysis of the red blood cells with Solution G1 (RBC Lysis Solution) was not complete.

The sample contains a higher than average number of Red Blood Cells(RBC). To lyse the cells completely, repeat the RBC lysis by adding 3 parts ofSolution G1 (RBC Lysis Solution) to 1 part of sample, incubate for 10 minutesat room temperature, then centrifuge as recommended in the manual for yourspecific protocol.

2. The white cell pellet is loose after centrifugation.

a. Increase the centrifugation time from 2 minutes to 5 minutes if working withlarge volume samples (15 ml or 50 ml tube).

b. The speed and time for the centrifuge might not be set correctly. Check the centrifuge settings. Please contact MO BIO’s Technical Services for help.

CELL LYSIS STEP

1. The cells isolated from a Ficoll gradient were not completely lysed.

The lysis was incomplete because the Ficoll was not sufficiently removed. Washcells with phosphate buffered saline (PBS) to remove the Ficoll before addingSolution G2 (Cell Lysis Solution). Incubate the lysate at 65°C to complete thelysis if needed.

2. The cells were not completely lysed.

a. The system is overloaded with cells due to the fact that the amount of SolutionG2 (Cell Lysis Solution) was insufficient compared to the amount of cells. TheCell Lysis Solution will become highly viscous and result in the clumping of cells.The lysis of cells can be completed by adding more Solution G2. To avoidincomplete cell lysis, count the number of cells using a hemacytometer or othercell counter based method before starting cell lysis.

b. Cells are not completely re-suspended before adding the Solution G2 (Cell LysisSolution) resulting in cell clumps. For lysing the cells in clumps, incubate thesample at 37°C with gentle mixing until the sample is homogeneous.Alternatively, add Proteinase K Solution to a final concentration of 100 µg/mland incubate the sample at 55°C (1 hour to overnight) to dissolve the cell clumpsat a faster rate and complete the lysis.

28

PROTEIN PRECIPITATION STEP

Problems with the protein pellet: soft, loose or no pellet

There are three different reasons why this may occur:

1. The centrifuge settings are not correct.

Make sure that the centrifuge speed is set to the recommended g-force called forin the protocol.

- If you are using microcentrifuge tube preps, set the centrifuge speed to maximum.

- If you are using a table top or any centrifuge other than a microcentrifuge, setthe speed to 2,500 x g. If this g-force can not be attained by the centrifuge,increasing centrifugation time can achieve the same total g-force. For example,if there is a requirement of 2,500 x g for 5 minutes for a particular protocol(25,000 x g = Total g-force x minutes) and the centrifuge only can achieve 1,000 x g, then it will be necessary to increase the spin time to 25 minutes.

RPM and g-force are two different units. Make sure to spin the samples at theappropriate speed. If not, you might get a lower yield, protein contamination oreven loose pellets.

Use the equation below to set your centrifuge to ensure the correct spinningspeed. g-force = 1.12 x r x (rpm/1000)2.

NOTE: r = radius of rotor in millimeters from center of rotor to center of tube cavity.

2. The cell lysate and Solution G3 (Protein Precipitation Solution) were not mixed properly.

Vortex as thoroughly as possible for 20 seconds as specified in the protocol.

3. A loose protein pellet is a sign that the sample was not cooled adequately (20-22°C) prior to the addition of Solution G3 (Protein PrecipitationSolution).

Before adding Solution G3, cool samples to room temperature or below. Followthese three steps to achieve a tighter protein pellet:

a. Repeat the vortexing for an additional 20 seconds to uniformly mix the ProteinPrecipitation Solution with the cell lysate.

b. Incubate the sample on ice for 10-15 minutes. (This should help in the formationof a tighter pellet.)

c. Pellet the precipitated proteins by centrifugation as mentioned in the protocol.

29

DNA HYDRATION STEP

Re-hydration of samples is taking longer then expected.

There are two possibilities:

1. Mixing of sample was not done properly during the hydration step.

Mix sample by gently tapping the sample tube to facilitate dissolving of DNA.

2. The DNA pellets were overdried before adding Solution G4 (DNAHydration Solution).

Samples that are dried for longer than 1-2 hours at room temperature or dried using a vacuum will need more time to re-hydrate completely. IncubateDNA samples at 65°C for an hour followed by incubation overnight at room temperature. Overnight incubation at 65°C is not recommended.

DNA QUALITY

1. The size of the DNA purified is 50 kb or less.

a. Inappropriate storage of the starting material may degrade DNA. Collect and store samples using methods that preserve DNA integrity.

- Short term storage (5 days): store at -80°C

Alternatively, the samples can be stored in Solution G2 (Cell Lysis Solution) at room temperature for extended periods of time.

b. The MO BIO protocol involves steps that produce a negligible amount of DNAshearing as compared to other traditional methods of DNA extraction. TheProtein Precipitation Step, which involves vortexing for 20 seconds, will not affect the size of the purified DNA fragments.

2. A260/A280 ratios obtained are too high or too low.

Extremely low or high concentrations of DNA will not give good A260/A280 ratios.For more accurate readings either concentrate by ethanol precipitation or trydiluting your sample.The expected A260/A280 ratios when using the UltraClean™Blood DNA Isolation kit range from 1.8-2.0. DNA quality can also be deter-mined by visualizing samples using agarose gel electrophoresis or byperforming a restriction digest analysis. Further evaluation can be done by performing PCR amplification using the DNA as a source of template DNA.

30

DNA YIELD

The DNA yield obtained is too low.

1. The number of cells in the starting material was too low. It is critical to use therecommended amount of cell material for a particular protocol. Count the number of cells prior to the Cell Lysis Step. In cases of insufficient numbers ofcells, the DNA concentration will be lower during the DNA precipitation stepand thus will reduce the DNA precipitation efficiency. A DNA carrier such as0.5 µl Glycogen (20 mg/ml) per 300 µl Isopropanol can be used to improve theefficiency of DNA precipitation if a low DNA yield is expected or if workingwith a small number of cells (

31

2. Invert several times to mix and incubate at 4°C for 30 minutes.

3. Centrifuge samples according to the following table:

4. Drain the supernatant, add 70% Ethanol (shown in the table on page 30) andinvert gently to wash the DNA.

5. Centrifuge at high speed as shown below.

6. Drain the supernatant and invert on a clean sheet of absorbent paper to allow theDNA to air dry for 15-20 minutes.

7. Once the DNA is dry, add the appropriate volume of Solution G4 (DNAHydration Solution) to the sample.

8. DNA can be stored short term at 4°C, but should be stored at -20°C or -80°Cfor long term storage.

Tube Speed Time2.0 ml tube 13,000 x g – 16,000 x g 10 minutes15 ml tube 2,500 x g 20 minutes50 ml tube 2,500 x g 20 minutes

Tube Speed Time2.0 ml tube 13,000 x g – 16,000 x g 10 minutes15 ml tube 2,500 x g 20 minutes50 ml tube 2,500 x g 20 minutes

OTHER QUALITY PRODUCTS AVAILABLE FROM MO BIO

LABORATORIES, INC.

Valuable supplies that can be used with this kit:

Product Description Catalog No.

2.0 ml microcentrifuge tubes (bag of 100) 1200-100-T

15 ml centrifuge tubes (bag of 25) 12700-T

50 ml centrifuge tubes (bag of 25) 12600-T

100% Isopropanol (2-propanol) (33 ml) 12000-100-5

70% Ethanol (30 ml) 12000-100-6

RNase A Solution 25 mg/ml (1 ml) 1202-1

RNase A Solution 25 mg/ml (5 ml) 1202-5

Proteinase K Solution 20 mg/ml (2 ml) 1222-2

DNA Isolation Kits

UltraClean™ BloodSpin™ DNA Isolation Kit (250 preps) 12200-250

UltraClean™ Mega BloodSpin™ DNA Isolation Kit (10 preps) 12210-10

UltraClean-htp™ 96 Well BloodSpin™ DNA Isolation Kit (4 x 96 preps) 12296-4

UltraClean™ Forensic DNA Isolation Kit (20 preps) 14000-20

UltraClean™ Tissue DNA Isolation Kit (50 preps) 12334-50

UltraClean-htp™ 96 Well Tissue DNA Isolation Kit (4 x 96 preps) 12996-4

PowerSoil™ DNA Isolation Kit (100 preps) 12888-100

PowerMax™ Soil DNA Isolation Kit (10 preps) 12988-10

PowerSoil-htp™ 96 Well Soil DNA Isolation Kit (4 x 96 preps) 12955-4

UltraClean™ Soil DNA Isolation Kit (50 preps) 12800-50

UltraClean™ Mega Soil DNA Isolation Kit (10 preps) 12900-10

UltraClean-htp™ 96 Well Soil DNA Isolation Kit (4 x 96 preps) 12896-4

32

33

RNA Isolation Kits

RNA PowerSoil™ Total RNA Isolation Kit (25 preps) 12866-25

UltraClean™ Microbial RNA Isolation Kit (50 preps) 15800-50

UltraClean™ Tissue RNA Isolation Kit (50 preps) 15000-50

DNA Purification Kits

UltraClean™ 15 DNA Purification Kit (300 preps) 12100-300

UltraClean™ GelSpin™ DNA Extraction Kit (100 preps) 12400-100

UltraClean™ PCR Clean-Up Kit (100 preps) 12500-100

Enzymes

Ribonuclease A (RNase A), 25 mg/ml Solution (1 ml) 1202-1

Ribonuclease A (RNase A), 25 mg/ml Solution (5 ml) 1202-5

Proteinase K, 20 mg/ml Solution 1222-2

Proteinase K, Lyophilized Powder (100 mg) 1223-100

OmniTaq™ DNA Polymerase Enzyme 1224-250

OmniTaq™ DNA Polymerase 2X Master Mix 1226-250

Omni KlenTaq™ DNA Polymerase Enzyme 1225-250

Omni KlenTaq™ DNA Polymerase 2X Master Mix 1227-250

Lab Supplies

Molecular Biology Grade Water (200 ml) 17012-200

UltraClean™ Lab Cleaner (1 L) 12095-1000

UltraClean™ Lab Cleaner (500 ml) 12095-500

Mini Horizontal Gel System 16001

Power Supply w/ Timer (110 V) 16023

Power Supply w/ Timer (220 V) 16023-220

RNase-Free Gloves (Small) 1555-S

RNase-Free Gloves (Medium) 1555-M

RNase-Free Gloves (Large) 1555-L

34

CONTACT INFORMATION

Technical information: Mon.-Fri. 8:00 AM to 5:00 PM (PST)

Evening and weekend inquiries may be left on voice mail at the numbers below or emailed to [email protected] for priority attention the following business day.

Phone:

MO BIO Laboratories, Inc.Toll Free 800-606-6246or 760-929-9911Fax: 760-929-0109Email: [email protected]

Mail:

MO BIO Laboratories, Inc.2746 Loker Ave WestCarlsbad, CA 92010

ORDERING INFORMATION:

Mon.-Fri. 8:00 AM to 5:00 PM (PST)

Direct:

Phone MO BIO Laboratories, Inc.Toll Free 800-606-6246 or 760-929-9911Fax: 760-929-0109Email: [email protected]

Mail:

MO BIO Laboratories, Inc.2746 Loker Ave WestCarlsbad, CA 92010

For the distributor nearest you, visit our web site at

www.mobio.com/distributors

35

NOTES:

36

NOTES:

UltraClean™ Blood DNA Isolation Kit · 2017. 11. 22. · 1 UltraClean™ Blood DNA Isolation Kit (PROCESSES 1,000 ml OF BLOOD) INTRODUCTION Use this kit for isolating high quality

Documents