In vitro transcription with T7 RNAP
aisoai at agc.co.jp
Fri Mar 7 12:07:09 EST 1997
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
2) You try to use RNase inhibitor during in vitro
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
>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
>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.
Atsushi Isoai, Ph.D. <aisoai at agc.co.jp | aisoai at yk.rim.or.jp>
Senior Staff Researcher, Research Center, Asahi Glass Co.LTD.
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