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
- 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!!!
- Mix gently; spin for 5
minutes at max speed.
- Remove aqueous layer
(top layer) to a new tube. Dump the bottom layer into phenol:chloroform
liquid waste container.
- Add equal volume of chloroform:IAA
(left cabinet under fume hood).
- Mix; spin 2 minutes at
- Remove aqueous (top) layer
to a new tube. Dump bottom layer into waste container.
B. Ethanol Precipitation
- Add sodium acetate to
- Add two volumes 100% ethanol
- Mix; spin 30 minutes at
- Remove supernatant carefully;
save to be safe (at least until you quantify to ensure that you didn’t
lose too much).
- Fill tube halfway with
70% ethanol; spin 2 minutes (this is a wash).
- Repeat wash.
- Carefully pipet out or
decant supernatant. There will be a clear pellet on the bottom. It
may be difficult to see.
- 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.
- 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.