Berglund:Phenol/Ethanol Extraction

From OpenWetWare
Jump to navigationJump to search

When cleaning up RNA with phenol, you want the pH to be low. Often, we buy (Ambion 9722) acid phenol pH 4.5 in order to do our extractions. For downstream applications (like enzymatic, protein related (binding or RT-PCR)) you will want to do the chloroform steps. For DNA, everything is the same except you want to use a buffered phenol (around 7.5):

Protocol for RNA phenol extraction with ethanol precipitation:

Increase volume of RNA to 300µl using water (reduces loss, it is better to work in a big volume).
Add 300µl phenol.
Vortex for 10 minutes.
Spin at high speed for 10 minutes.
Remove only 280µl of the upper layer (this is the aqueous phase). Do not touch the interface, it is full of denatured protein.
Place aqueous phase in a fresh tube.
Add 300µl of chloroform.
Vortex for 10 minutes.
Spin at high speed for 10 minutes.
This removes residual phenol. Sometimes you do a second chloroform extraction to make sure you have removed residual phenol.
Place 260µl of aqueous phase into a fresh tube.
Add 26µl 3M sodium acetate pH 5.2 to the tube.
Add 1µl of glycogen if you would like (it is a carrier, sometimes you won't want a carrier in your downstream applications)
Mix well.
Add 715µl of 200proof ethanol.
Mix well.
Freeze at -20°C for 1 hour or longer (if you freeze at -80°C it often doesn't precipitate as well, it is better to do it at -20°C)
Spin for 10-20 minutes in the cold at high speed.
Remove the supernate (ethanol and salts)
The pellet should be visible at the bottom.
Spin briefly.
Remove remainder of ethanol carefully.
Allow pellet to dry for 2 minutes (if it dries fully it will stick to the wall of the tube)
Can be stored at -80 or in -20 as a pellet until needed.

See excellent reviews (some talk about using Ammonium acetate vs. Sodium acetate- which is useful if you are working with small RNAs):

update 8/15/06- Kristy

Something I just read in the Ambion catalog, which may be informative / useful to one or more of you:

"Measurement of phenols with pH paper is not accurate due to breakdown of the indicator chemicals on the paper. To measure pH of phenol solutions with a pH meter, mix 2 mL of the organic phase with 8 mL of methanol and 10 mL of H2O. Measure the pH of the total sample with a standard pH meter."