1. Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to your sample, and vortex
thoroughly for approximately 20 conds, solution should be milky.
feger
phenol (removes protein)
Chloroform (removes phenol)
100% isopropyl alcohol (precipitates DNA)
75% Ethanol (washes out salt)
2. Centrifuge at room temperature at 13,000 rpm for 2 minutes. Carefully remove the upper
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aqueous pha, and transfer the layer to a fresh tube. Be sure not to carry over any phenol during pipetting
3. Proceed to isopropyl alcohol Precipitation. Add 0.6 volume of isopropyl alcohol to the
aqueous pha, mix thoroughly and incubate sample at -20℃for 30 min.
4. Centrifuge the sample at 4°C for 15 minutes at 13000rpm to pellet the cDNA.
remain的用法5. carefully add 1 mL cold 75% Ethanol (do not vortex).
6. spin 2 minutes at 12000 rpm at 4°C.
7.carefully pour out / aspirate supernatant (do not lo DNA-pellet)
六级分值分配8. carefully add 1 mL cold 75% Ethanol (do not vortex).
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9. spin 2 minutes at 12000 rpm at 4°C
10. air dry 10 minutes at room temperature (do not overdry, becau DNA becomes hard to dissolve)
arnal钾霞石11.dissolve in:
10 mM Tris pH 7.5 (+++)
smoking gunTE-Buffer (++) - EDTA may inhibit downstream enzymatic reactions
ddH2O (+) - freeze at -20°C becau unbuffered DNA undergoes degradationhalogen>曾几