RNA Probe woes

Tracy Aquilla aquilla at salus.med.uvm.edu
Mon Sep 19 09:07:31 EST 1994


In Article <35jnec$lk9 at cc.tut.fi>, bljulu at uta.fi (Jukka Luoma) wrote:
>D.Greeve <DAEMON> (darreng at ccmar.csiro.au) wrote:
>: Hi netters.
>
>:    I am currently experiencing difficulties in producing P32 labeled RNA 
>: probes from a "Bluescript KS+"  plasmid. I am currently using a kit marketed 
>: by "Bresatec" (- an Australian company), designed to do this, but I am
getting 
>: almost no transcription using T7 and my plasmid. The kit control appears to 
>: work, however with identical conditions, my plasmid does not.
>
>: Any ideas on the matter would be much appreciated....
>
>: Darren Greeve.
>
>I have had similar problems and usually the cause of the promlems was too
>low plasmid concentration. Are you sure about your plasmid concentration?
>Your plasmid concentration should be about 0.1ug/ul in the final volume.
>
>Jukka


This is often the case when the probe template or transcript itself have the
ability to form stable secondary structures. Certain templates just do not
transcribe efficiently in vitro. You can test this by using a program to
check for potential secondary structures in the template and transcript. If
secondary structures are indicated, you may need to find another region to
use as your probe. Good luck.

Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu



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