Richard M. Rohan rrohan at umabnet.ab.umd.edu
Tue Jun 27 20:24:50 EST 1995

On Wed, 21 Jun 1995, Tracy Aquilla wrote:

> In Article <1995Jun19.100817.54281 at cc.usu.edu>, sl6dp at cc.usu.edu wrote:
> >Does anybody know if there is a minimum length for a good riboprobe?
> >I am thinking of making 300bp max. riboprobes, is there any drawbaks for 
> >this, any special meassures to consider???
> I routinely make riboprobes over 500 bp with no problems. Longer probes will
> give greater sensitivity in an RNAse protection assay, but can give some
> problems if the RNA being assayed is somewhat degraded.
>     tracy
I also use riboprobes up to 500 nt in the RNase protection assay.  
(Shortest riboprobe is 90 nt).  One drawback in the use of long probes is 
the difficulty of synthesizing full-length probe of high specific
activity (ie 50 microCi 32P-UTP, 800Ci/mmole, no unlabelled UTP). I do T7
transcriptions at room temperature to prevent premature termination (I think
that I found this idea in an Ambion manual).  Also, gel purification of the
full-length probe prevents background smearing from short transcripts and
decreases the amount of radioactivity needed in the RPA.

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