DNA?Precipitation

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 .

dH₂O (+) - freeze at -20℃because unbuffered DNA undergoes degradation .