RNase removal by phenol-chloroform ?

nierlich at ucla.edu nierlich at ucla.edu
Wed Dec 4 03:02:56 EST 1996

In <57utfc$69g at mserv1.dl.ac.uk>, deepak at bgumail.bgu.ac.il (Deepak Khandka) writes:
>Is  it possible to remove RNase contamination from a Tris-containing buffer
>by extracting with phenol-chloroform without purturbating the pH of the
>buffer ?
>Deepak K Khandka
>Ben-Gurion University

If properly prepared, the phenol phase of your p-c, should have been pre-equilibrated with an appropriate buffer; e.g.,
something close to the pH of the solution that you wish to extract.  Otherwise it will change the pH, depending on its
age, oxidation, etc.

One way to make your buffer RNase-free is to treat with diethylpyrocarbonate, as described in Maniatis.  Add (I think) 
0.1 % DEPC to the buffer, mix, invert the bottle with a slightly loose cap so the neck is wetted, let stand for a few 
hours--overnight, autoclave (to drive off residual DEPC/alcohol/CO2), and store until needed.

Don Nierlich

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