ISOLATION
OF GENOMIC DNA FROM TISSUE CULTURE CELLS
Resuspend cells well and count.
Pipet cells to be analyzed into a conical tube and centrifuge (250Xg, 5min).
Aspirate the culture medium. Wash the cells (2X) with PBS/CMF (calcium and
magnesium free). On the final wash, aspirate the supernatant.
Resuspend the cells in 19ml STE
buffer. Add 500m l of 20% SDS to lyse the cells. Upon mixing
the solution should become visibly viscous. Add 500m l
of 10mg/ml Proteinase K and incubate the lysate overnight at 37° C.
Extract the denatured proteins by
adding 25ml of Tris-HCl (pH 8) saturated phenol. Mix the emulsion for 30
minutes on a Nutator or similar mixing platform. Place the tube on ice for ten
minutes to allow the two phases to separate. Spin the emulsion for 10 minutes
at 2400rpm to obtain a good separation of the two phases.
Pipet the upper, aqueous phase into
a fresh 50ml conical tube. Extract with 25ml of chloroform (HPLC grade):Isoamyl
alcohol (24:1). Mix 30 minutes as before. Place the emulsion on ice for ten
minutes, then centrifuge for ten minutes at 2400rpm.
Pipet the upper aqueous layer into
a fresh 50ml conical tube. Pour the entire contents into a plastic flask
containing 200ml of absolute ethanol. The DNA should begin to precipitate
immediently, but allow the solution to stand at room temperature for several
minutes. The precipitate will condense into a small ball of DNA. Pick the DNA
out of the ethanol with a Pasteur pipet and place it in a clean microcentrifuge
tube.
Wash the DNA 3X with 85% ethanol.
Spin briefly each time in a microcentrifuge, then aspirate the supernatant. Dry
the DNA in the Speed-Vac and resuspend in an appropriate volume of 10mM
Tris-HCl pH 7.5/1mM EDTA. Allow the DNA to dissolve overnight at 37° C.
If the DNA is too concentrated,
aliquot the DNA into another tube and add Tris/EDTA. Dilute the DNA 30x (20m l
DNA in 600m l water). Read the A260.
Calculate the DNA concentration:
A260 x Dilution x 40 = m g
DNA/ml
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Solutions: |
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STE (per liter) |
0.1M NaCl 0.05M Tris 1mM EDTA Deionized water Adjust pH to 7.4 with HCl Store at 4° C |
5.844 grams 6.057 grams 0.3722 grams |
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Proteinase K solution |
25mg Proteinase K (Protease, type X1, fungal, Sigma #P-0390) is dissolved in 2.5ml of 10mM Tris-HCl pH 7.4. Store at -20° C. (10mM Tris is 1.2114g/l, adjust pH with HCl to 7.4) |
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Phenol saturated with 200mM Tris |
Phenol redistilled into 200mM Tris (200mM Tris is 24.22g/l. Adjust pH to 8.5 with HCl) Place the phenol in a one liter flask on a magnetic stirrer. Add the Tris with stirring while measuring the pH. Stir until the pH equilabrates. Stop the stirrer and allow the two phases to separate. Aspirate the Tris from the flask and add fresh Tris. Stir until the pH equilibrates. Continue this process until the pH reaches approximately 8.5. Add 0.1% hydroxyquinoline per liter of phenol only as a preservative. Store in a brown glass container. |
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Chloroform-isoamyl alcohol |
24 parts Chloroform to 1 part isoamyl alcohol by volume |
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Ref: Poncz,M., D.Salowiejczyk, B.Harpel, Y.Mory, E.Schwartz, and S.Surrey (1982). Construction of human gene libraries from small amounts of peripheral blood: Analysis of b -like globin genes. Hemoglobin 6(1):27-36. |
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