AMBIONS ANTISENSE 18S TEMPLATE

Bernard Murray bernard at elsie.nci.nih.gov
Tue Aug 20 12:18:28 EST 1996


In article <32190cd11b0b002 at mhub0.tc.umn.edu>, cause001 at MAROON.TC.UMN.EDU 
says...
>
>I have been having trouble with the T7 18s transcribed probes from Ambion 
>on RPAs.  I can not seem to: 
>
>(a)   get the correct specific activity so it will show up but not so much 
>so it wont overexpose.
>
>(b)   transcribe enough probe to saturate the RNA sample (it does not 
>titrate)

I played with this template before moving to their cyclophilin control
template which requires much less work - I don't know if any of their
offerings are sufficiently homologous with porcine cyclophilin.  This
also has the advantage that you can boost the sensitivity by using
polyA-enriched RNA.

Anyway, my approach with the 18S was essentially as they recommend in
the instructions.  I used their MegaShortscript kit and performed a
large transcription in the absence of radionucleotides to generate
a stock of cold transcript which I could then keep for some time.
For routine assays I performed a relatively small scale transcription
with 32P-NTP (either UTP or CTP) and then diluted the product with the
cold transcript.  It takes a few goes to get the ratio correct but then
there are no problems.  Although Ambion claim you don't have to clean up
the transcript from the 18S before performing RNase protection I actually
found this to be a helpful step as I could then count a little of the
product and this helped me to decide the ratio of hot/cold.  I used
an acrylamide gel but I've heard success with people using spin columns
to remove the bulk of the ribonucleotides.
	I had always wondered if it was possible to perform a standard
transcription with highly diluted 32P-NTP but your results suggest that
this may not be the best approach.
	Good luck,
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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