1.Prepare or obtain Buffered phenol,pH 8.0.Add 2 crystals of 8-Hydroxyquinoline to prevent oxidation.This also identifies the organic phase as yellow-colored. 2.Combine DNA sample with an equal volume of Buffered Phenol. 3.Mix well by vortexing until uniformly milky-yellow. 4.Centrifuge for 10 min at 4℃. 5.Promptly remove upper aqueous phase and transfer into a new tube.DO NOT remove the interface. 6.Add an equal volume of 24:1 Chloroform:Isoamyl alcohol. 7.Mix well by shaking the tubes.Vortexing does not help. 8.Centrifuge for 3 min at 4℃. 9.Remove supernatant and transfer to a new tube. 10.For small amounts of DNA,add 2 ml 1% linear Polyacrylamide carrier for each 100 ml of sample. 11.Mix well.Add 2.5 volumes of 95% ethanol.Mix by inversion. 12.For small amounts of DNA,incubate at -20℃ for 30 min.Centrifuge for 15 min.For large quantities of DNA (e.g. chromosomal preps),centrifuge immediately for 3 min. 13.Remove supernatant.Add a large volume of 70% ethanol.Mix well to dislodge the pellet. 14.Centrifuge for 2 min.Remove the supernatant and air dry. 15.Resuspend DNA in water or TE. |
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