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        DNA Precipitation

        互联网

        1108

        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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