DNA Purification and Precipitation

1.Prepare or obtain Buffered phenol,pH 8.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℃hloroform: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.