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Termination of SP6 RNA polymerase during mRNA production

Cloning in vitro transcrip SP6 polymerase

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#1 StephanK82

StephanK82

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Posted 08 September 2011 - 12:58 PM

Hi!

I am currently generating a vector system to assemble ORFs. I include an SP6 site 5' and a pA site 3' of my gene of interests, so I can quickly generate mRNA using the SP6 polymerase. I know, usually the vector is cut behind the pA and a run-off transcript is generated. However, since I would like to make that somewhat large-scale I would like to skip the restriction digest step. Furthermore, I only have limited restriction sites available which might be a problem if such a site is in my gene.

I was wondering if you know if a normal termination signal that is used in bacteria (like these small hairpin loops) could be included after the pA. It has to be strong so that the SP6 polymerase in a mMessage mMachine reaction surely falls off. Then I would not need to digest the vector beforehand and still would get proper mRNA. Do you know if such a signal exists?


Thank you very much in advance!

all the best,
Stephan





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