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
