RNA synthesis

Laura K. Moen, Ph.D. LKM100F at ODUVM.CC.ODU.EDU
Fri May 6 10:34:55 EST 1994

Hi, Netters!
   We are having difficulty synthesizing labeled RNA to use as a substrate for
RNAse H assays after annealing with a complementary DNA.  We have made
unlabeled RNA before, and we routinely recover 25-30ug in a 20ul reaction run
under identical conditions.  However, if we add our labeled substrate to these
reactions, we get a 50-fold decrease in the yield.  We quantify the amount
of RNA by A260 readings and/gel electrophoresis.
   One idea we have is that the label might be contaminated with RNases...
this is a possibility.  However, we are using Tritiated ATP, and we have
also heard that ATP incorporation doesn't work well.  While this might be a
problem for making labeled probes at high specific activity, I am not sure that
the actual RNA yield would drop so dramatically.  So, does anyone have any
suggestions?  I would really appreciate hearing from someone who has done the
labeled synthesis successfully - thanks a lot in advance.  Laura Moen
EMail LKM100F at ODUVM.cc.odu.edu  (United States)

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