RNase removal by phenol-chloroform ?

Steven Cohen sbc4 at PO.CWRU.EDU
Wed Dec 4 09:21:02 EST 1996


At 08:02 AM 12/4/96 GMT, you wrote:
>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
>>Israel
>>-------------------------------
>
>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
>>
>
My best recollection is that chemicals containing primary amines, like TRIS,
should not be treated directly with DEPC. Better to treat the H2O and then
dissolve TRIS(which has been handled with care, no fingers!).

Steve




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