*Reliable* RPA method?

Pamela Norton pnorton at lac.jci.tju.edu
Tue Jul 16 17:39:09 EST 1996


In article <4scc5s$hqi at hobyah.cc.uq.oz.au>, hardy at biosci.uq.edu.au (Phil
Hardy) wrote:

> For the past year I have been using a commercial RPA kit and have
> become dissatisfied with the seeming unreliablity of the one-step
> RNase inactivation/precipitation procedure (no, it's not an RNase
> contamination problem).  
> I'm on the lookout for a more robust two-step RPA procedure and have
> re-started at the grass-roots level, Current Protocols in Molecular
> Biology.
> If anyone has some tips or modifications they would like to share with
> me, I would be most grateful.
> 
> Thanks in advance,
> Phil Hardy
> 
> hardy at biosci.uq.edu.au

   The following has proven very reliable:

   After RNase treatment (in about 250 ul volume) adjust to 0.5% SDS and
add 5 ul of 20 mg/ml Proteinase K. Incubate 15 minutes, 37oC. Do a single
extraction with phenol-chloroform, then ethanol precipitate, adding
appropriate amounts of NaAC, a little EDTA and a few ug of yeast tRNA as
carrier. After centrifugation, remove supernant _completely_ (or do EtOH
wash) and resuspend pellet in formamide containing gel loading mix.

   It is possible to omit the protease step if one titrates the amount of
RNases and uses the minimum amount needed to degrade the unhybridized
probe to smallish fragments. I might also add that I add the mixture with
the RNases in it to the hybridization mixture and mix by pipetting up and
down a few times, not vortexing, so as to prevent the enzymes from
spreading all over the tube and potentially carrying through to later
steps. 

   Hope this helps,

      Pam Norton

-- 
Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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