in vitro transcription

Marie Termansen M.Termansen at immi.ku.dk
Mon Sep 28 07:35:39 EST 1998


Hello

I am working with in vitro transcription and I have some problems with
getting rid of the template DNA after the in vitro transcription. In the
protocol they recommend to treat the sample (100 ul) for 10 min. with 5U
RNase free DNase. I did that and I even tried to treat the sample for 2
hours and there were still DNA left. (analyzed by running PCR on the
sample without adding reverse transcriptase). I have heard that some DNA
polymerases have RT activity, but since I haven't observed this
phenomenon in any of my previous reaction where I use the same DNA
polymerase I find it hard to believe that this can be the explanation. I
use 5 ug of DNA template which is a lot, but that is what they recommend
in the protocol. I will be very pleased if anyone can help me with my
problem.

Marie Klinge Termansen
IMMI, University of Copenhagen




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