从新鲜或冷冻组织中提取DNA

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_____________________________________________________________Volume in μlTotal           400 800 1200 1800 3200 4800 6400 8000DNA buffer 360 680 1020 1360 2720 4080 5440 6800Proteinase 20   40    60       80     160  240      320 40010% SDS   40    80   120    160    320   480    640  800