Reagents Chloroform Preparation DNA buffer(Tris-EDTA) 1 M Tris pH 8.0 20 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 |
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