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