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 
RNA polymerase? 

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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