Phenol (removes protein)
1.add equal volume of Phenol (= tris-saturated Phenol-Chloroform-Isoamyethanol)
2.vortex
3.spin 2 minutes at 12000 rpm 4℃
4.transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)
Chloroform (removes phenol)
1.add equal volume of Chloroform
2.vortex
3.spin 2 minutes at 12000 rpm 4℃
4.transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)
100% Ethanol (precipitates DNA)
1.add 0.1 volume 3 M sodium acetate
2.add 2.5 volumes 100 % Ethanol
3.vortex
4.precipitate at:
-20℃overnight ( )
-80℃1 h ( )
dry ice 15min ( )
5.spin 20 minutes at 12000 rpm 4℃
6.carefully pour out / aspirate supernatant (do not lose DNA-pellet)
70% Ethanol (washes out salt)
1.carefully add 1 mL cold 70% Ethanol (do not vortex)
2.spin 10 minutes at 12000 rpm 4℃
3.carefully pour out / aspirate supernatant (do not lose DNA-pellet)
4.air dry 10 minutes at room temperature (do not overdry, because DNA becomes hard to dissolve)
5.dissolve in:
10 mM Tris pH 7.5 ( )
TE-Buffer ( ) - EDTA may inhibit downstream enzymatic reactions .
dH2 O ( ) - freeze at -20℃because unbuffered DNA undergoes degradation .
