T7 in vitro transcription

Vayuputra vayuputra369 at hotmail.com
Sat Jul 19 13:01:57 EST 2003


Hello,

I made a couple of constructs with pBlueScript SK+ as the backbone to use in
in vitro transcription reactions (capped mRNA synthesis using the mMessage
kit from Ambion) with T7 RNA Polymerase.  I always had problems and never
could make any RNA.  Recently I realized that in the many subcloning steps
involved in adding a myc epitope (from pCS2+ vector) to the 3' end of the
gene/construct a second T7 promoter got cloned downstream of the gene.  So,
the construct looks like this:

T7 promoter -- gene -- myc epitope -- T7 promoter -- polyA

Can anyone tell me if the downstream T7 is the reason I never got RNA made
in my in vitro transcription reactions?  I sincerely appreciate all comments
and any suggestions in this regard.

Thanks a lot.
V





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