RNAse protection without phnole/chloroform

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Mon Nov 1 11:40:41 EST 1999


In article <oberstra-0111991509310001 at gen-pc2.biologie.uni-kassel.de>,
oberstra at hrz.uni-kassel.de (Juergen Oberstrass) wrote:
> I want to perform RNAse protection assays. In the normal protocol
> you have
> to digest the RNAse with protease and extract with
> phenole/chloroform to
> get rid of active RNase after the digestion step.
> Now we have problems with the organic radioactive waste and I
> would also
> like a faster and more convenient procedure. Ambion sells a kit
> which
> contains a inactivation/precipitation solution. Does anybody know
> the
> composition of this solution (I suppose it contains a chaotropic
> salt and
> a reducing agent) or has another good alternative for the
> proteinase K
> step?

Have you tried just precipitating after Proteinase K digestion without
phenol extraction? The RNAse shoould be dead after PK, and the carry
over of the PK shouldn't hurt anything downstream.

BTW, is the problem with phenol in general, or radioactive phenol? If
the latter, can't you just hold it until it decays to background (I'm
assuming you're using 32P here), then treat it like ordinary phenol
waste?

Nick



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