DNA Preparation from Fresh/Frozen Tissue

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 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