phenol contamination - (Aug/22/2005 )
After using the QIAamp virus RNA extraction kit then Nanodrop to measure the integrity of my RNA, I always obtain very high 260/280 ratios (> 3). Unfortunately, I did not record the absorbance at 230 nm which, after reading a few posts in this forum, seems indicative of the amount of organic contamination present. Since the volumes I work with are very small, I wonder if people have had luck in boosting up the sample volume to about 200 ul (as suggested previously in the forum) with RNase-free water before extracting with phenol:chloroform in terms of minimizing organic contamination? Any suggestions on how to get rid of any remaining contaminating phenol still present after taking off the aqueous layer?
To remove phenol, extract with chloroform once or twice, followed by an ethanol precipitation. If you have small amounts of DNA or RNA then consider a co-precipitant such as tRNA, glycogen or Novagen pellet-paint.
Since you have to ethanol precipitate in the end, I always increase the volume of my organic extrations to 400ul. That way 2x vol of ethanol and 0.1x vol salt fits neatly into a 1.6ml eppendorf tube.