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| 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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