In vitro transcription with T7 RNAP
Sami K J Kukkonen
skjkukko at cc.Helsinki.FI
Tue Mar 4 08:17:44 EST 1997
Has anyone else faced problems with in vitro RNA transcription with T7
I'm getting very faint or smeared RNA bands with the system. I've had
this problem before and it seems to be due to the impurity of the DNA
template. Of course it might be due to something else as well.
I need to do restriction before the RNA transcription. Have you got any
advice what DNA purification method would yield pure enough DNA that I
would get rid of this problem?
So far I've tried 1) phenol-chloroform extraction without subsequent ether
treatment, 2) Geneclean II, 3) Qiagen's Qiaquick PCR purification. None
of these give me consistently pure DNA. They work sometimes for the RNA
transcription and sometimes they don't.
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