run-off transcription

[brett]

>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).


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu