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°C.

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°C.

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°C 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°C;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°C.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°C 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°C.

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

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°C.

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°C.

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°C on a rotating shaker.

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