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.