brett at BORCIM.WUSTL.EDU
Tue Mar 25 21:42:00 EST 1997
>I've read several times that it is preferable to linearize my vector for T7 in
>vitro transcription with a UNIQUE restriction site. Does this mean the entire
>plasmid should have only one of the required sites (to terminate the
>transcript)? Or does this mean to make sure that your desired transcript
>sequence does not contain this site? If the entire plasmid should have only
>one of the linearization sites, why is this necessary? Do extraneous
>linear DNA fragments somehow inhibit T7 pol from recognizing the T7 promoter?
> Any advice would be greatly appreciated.
> Thank you
> Sean Rollins
> Rollins.24 at osu.edu
You're right the first time. The RE can cut a million times, as long as it
doesn't cut in your desired template DNA. Note that nicked DNA can give funky
products, and 3'overhangs are most likely to give you contaminating (-) strands
(relative to your product).
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu
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