I am using the T7 Polymerase to in vitro transcribe mRNA using the Ambion mMessage mMachine Kit. For this I linearize a plasmid and the reaction works really great. My question concerns how the end of my RNA will look like. I use a restriktion site directly behind the stop codon for which I am making mRNA. So I have .....TGACTCGAG...., cut with XhoI and will end up with ....TGAC The concern that I have now is, that after the stop codon TGA, there is only one base left. Will the T7Polymerase transcribe the template until the very end, or will the last few nucleotides be missing. Thus: Will all the mRNA have TGACAAAAAAAAAAA..... after tailing or will there be many that do not have the stop codon.
I hope there is some help out there,
thanks in advance,
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in vitro transcription template
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