RPA/Andrew Devitt

Richard M. Rohan rrohan at umabnet.ab.umd.edu
Tue Feb 7 13:00:53 EST 1995


> In <devitta-0302951029270001 at bcs93.bham.ac.uk> devitta at uk.ac.birmingham writes:
> 
> > In article <Uphoff-0102951549120001 at jmm2.ahabs.wisc.edu>,
> > Uphoff at ahabs.wisc.edu (Tim Uphoff) wrote:
> > 
> > >    I am looking for some advice regarding RPAs.
> > > Specifically we are trying the Ambion RPAII kit and having difficulty
> > > obtaining complete digestion of the transcript with nonspecific or no RNA
> > > control samples.
> > > 
> 	If I understand this thread correctly, you folks can't completely rid
> yourselves of the probe following the RNAse digestion.   There is a simple 
> explanation that is quite logical that may help.  Is it possible that there is
> plasmid template left over from the transcription  reaction?  If so, it is a
> dandy candidate for the hybridization of your probe!  Like, total
> complementation, dude!  As this hybrid is a double stranded duplex, it is
> immune from digestion and will appear as "undigested probe".  A solution is to
> extend the DNAse I digestion period, or try some new DNAse I (fresh) to rid
> yourself of this target.  
> 
Another source of totally complementary sequences can be sense strand 
transcripts.  These will not be eliminated by extending the DNase step.  
These sense strand transcripts can arise from non-specific transcription 
from non-linearized plasmid templates or from templates which have been 
linearized with restriction endonucleases that leave blunt or 3' 
overhangs.  Be sure that you are using a 5' overhang enzyme to linearize 
your template, that the digestion is complete and, if necessary, gel 
purify the linearized DNA prior to transcription.  Using two different 
enzyme for the linearization step can also help.  In our experience 
(which is extensive) gel-purification of the riboprobe also helps 
eliminate this problem.  Finally, only add enough riboprobe to be in 10X 
excess for your target.  Adding more will only increase the non-specific	
background.
Hope this helps.
Rich Rohan
University of Maryland
rrohan at umabnet.ab.umd.edu



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