In vitro transcription with T7 RNAP

[ISOAI Atsushi]

Hi, Sami
I had also tried to synthesize RNA in vitro using
T7 RNA polymerase.I used Invitrogen "in vitro 
transcription kit" and carried out a series of 
experiment. (It worked well.)  After restriction 
enzyme reaction and protease treatment, I purified 
DNA by conventional method, that's phenol-chloroform
extraction method. There were no problem. 
My suggestions are:
1) Please try QIAGEN plasmid isolation Kit for preparing
template DNA. We usually use RNase to isolate plasmid 
DNA from E.coli. I think it is difficult to remove RNase
by PEG-precipitation method. QIAGEN plasmid isolation Kit
works well.
2) You try to use RNase inhibitor during in vitro 
transcrition reaction.
3)Finally I recommend you to handle template DNA as 
RNA (!!) just after final step of plasmid isolation.


In article <5fh7do$fbi at oravannahka.Helsinki.FI>, Sami K J Kukkonen
<skjkukko at cc.Helsinki.FI> wrote:

>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. 
>-- 
>Sami

=================================================================
Atsushi Isoai, Ph.D. <aisoai at agc.co.jp | aisoai at yk.rim.or.jp>
Senior Staff Researcher, Research Center, Asahi Glass Co.LTD.
URL: http://www.yk.rim.or.jp/~aisoai/index.html
=================================================================