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 |
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