DNA Purification - Extraction 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 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 µL 1% linear Polyacrylamide carrier for each 100 µL 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.