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