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DNA Purification - Extraction and Precipitation

Jeff Lawrence (P31)

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 C.

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 C.

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 C 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.