Phenol-Chloroform Extraction


Purpose: Phenol-chloroform extractions are done when you need to purify DNA from a solution that also has proteins. The DNA will dissolve in the aqueous layer, and everything else will go into the non-aqueous layer. If you simply need to concentrate your DNA, or need to change the buffer, perform only the ethanol precipitation portion.


**Be careful when working with phenol:chloroform!! Wear gloves and use fume hood. Dispose of waste in appropriate waste containers: tubes and tips in phenol:chloroform tubes/tips container; liquid waste in phenol:chloroform container.


A. Phenol:chloroform

  1. Add one equal volume of phenol:chloroform:IAA (found in 4C in room 263) to DNA solution. The phenol:chloroform solution has two layers – be sure to take the bottom layer!!!
  2. Mix gently; spin for 5 minutes at max speed.
  3. Remove aqueous layer (top layer) to a new tube. Dump the bottom layer into phenol:chloroform liquid waste container.
  4. Add equal volume of chloroform:IAA (left cabinet under fume hood).
  5. Mix; spin 2 minutes at max speed.
  6. Remove aqueous (top) layer to a new tube. Dump bottom layer into waste container.


B. Ethanol Precipitation

  1. Add sodium acetate to 0.3M
  2. Add two volumes 100% ethanol
  3. Mix; spin 30 minutes at 4C
  4. Remove supernatant carefully; save to be safe (at least until you quantify to ensure that you didn’t lose too much).
  5. Fill tube halfway with 70% ethanol; spin 2 minutes (this is a wash).
  6. Repeat wash.
  7. Carefully pipet out or decant supernatant. There will be a clear pellet on the bottom. It may be difficult to see.
  8. Dry the pellet by placing the tube upside down on a rack. It shouldn’t take longer than 30 minutes – just until all residual ethanol has evaporated.
  9. Dissolve pellet in appropriate amount of TE or desired buffer.

*If, after quantifying DNA solution, the concentration is lower than you expected, spin down the saved supernatant from step 4 again, starting at step 3.