in vitro transcription/translation

brett brett at BORCIM.WUSTL.EDU
Tue Jul 28 08:56:29 EST 1998


>Dear friends,
>
>I'd like to do some coupled in vitro transcription/translation assay
>from PCR products. My template cDNA is in pAD-GAL4, an activation domain
>two-hybrid vector from Stratagene. Unfortunately, this vector has a T7
>promotor downstream of the insert, so it is of no use for my purposes.
>That's why I thought to try a PCR approach with a SP6 promotor included
>into the forward primer, but I am not sure about the reverse primer.
>Has it necessarily to be placed within the ADH1 terminator so that it
>contains some sort of termination signal,or does the polymerase kind
>of fall off the fragment anyway and the only thing necessary is a
>stop codon for the translation?
>Any hints would be appreciated.
>
>Cheers
>Ricky

Yes, SP6 will "fall off" the template, producing a run-off transcript.  This
should be translatable, assuming proper signals are present, such as a 5' cap
structure or IRES element.  Translation does appear to be affected by sequences
at the 3' end, so you may want to consider this when designing your template.
I highly doubt a nuclear polyA signal will do you much good though.  Best of
luck.

Brett Lindenbach
brett at borcim.wustl.edu
Dept of Molecular Microbiology
Washington University School of Medicine

"I have never let my schooling interfere with my education. "
     - Mark Twain





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