Section of Cancer Genomics,Genetics Branch,NCI

National Institutes of Health

Reagents

Chloroform

EDTA,0.5 M

Ethanol,absolute

Isoamyl alcohol

Sigma,Cat.I-3643

Phenol

Phosphate Buffered Saline (PBS),1X

Proteinase K

EM Science,Gibbstown,WV Cat.24568-2 (100 mg)

RNase A

Boehringer Mannheim,Cat.109 169

Sodium dodecyl sulfate (SDS)solution,10%

Digene Diagnostics,Beltsville,MD,Cat.3400-1016

Preparation

DNA buffer (Tris-EDTA)

1 M Tris pH 8.0 20 ml

0.5 M EDTA 20 ml

Sterile water 100 ml

Proteinase K (10mg/ml)

Dissolve 100 mg Proteinase K in 10 ml TE for 30 min at room temperature (RT)

Aliquot and store at –20℃

RNase A (20 mg/ml)

Dissolve 200 mg RNase A in 10 ml sterile water,boil for 15 min,and cool to RT.

Aliquot and store at –20℃

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Procedure

1.Put 60-80 mg of tissue in a petri dish with culture media and divide the tissue into two pieces.

2.Put the tissue into two sterile 15 ml tubes and centrifuge for 2 min at 4℃ at 1500 rpm.

3.Remove the supernatant,and wash twice with 1 ml 1X PBS or DNA-buffer.

(It is possible to store the pellet at -80℃; in that case,add 1 ml 1X PBS and resuspend the pellet.Use a cryo-tube and centrifuge at 1500 rpm for 2 min at

4℃.Remove the supernatant,and freeze the pellet.)

4.Remove supernatant and resuspend the pellet in 2.06 ml DNA-buffer.

5.Add 100 μl proteinase K (10 mg/ml)and 240 μl 10% SDS,shake gently,and incubate overnight at 45℃ in a waterbath.

6.If there are still some tissue pieces visible,add proteinase K again,shake gently,and incubate for another 5 hr at 45℃.

7.Add 2.4 ml of phenol,shake by hand for 5-10 min,and centrifuge at 3000 rpm for 5 min at 10℃.

8.Pipette the supernatant into a new tube,add 1.2 ml phenol,and 1.2 ml chloroform/isoamyl alcohol (24:1); shake by hand for 5-10 min,and centrifuge

at 3000 rpm for 5 min at 10℃.

9.Pipette the supernatant into a new tube,add 2.4 ml chloroform/isoamyl alcohol (24:1),shake by hand for 5-10 min,and centrifuge at 3000 rpm for 5

min at 10℃.

10.Pipette the supernatant into a new tube,add 25 μl 3 M sodium acetate (pH 5.2)and 5 ml ethanol,shake gently until the DNA precipitates.

11.Take a glass pipette,heat it over a gas burner,and bend the end to a hook.Fish the DNA thread out of the solution using the hook and transfer DNA to a new tube.

12.Wash the DNA in 70% ethanol and dry it in the speed vac.

13.Dissolve the DNA in 0.5-1 ml sterile water overnight (or longer if necessary) at 4℃ on a rotating shaker.

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14. Measure the DNA concentration in a spectrophotometer and run 200 ng on a 1% agarose gel.

Tissue (mg) 5 10 15 20 40 60 80 100
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Volume in μl
Total 400 800 1200 1800 3200 4800 6400 8000
DNA buffer 360 680 1020 1360 2720 4080 5440 6800
Proteinase 20 40 60 80 160 240 320 400
10% SDS 40 80 120 160 320 480 640 800