In vitro transcription addendum

the End jgraham at bronze.ucs.indiana.edu
Sun Dec 5 10:18:24 EST 1993


Apparenlty in some instances templates with regions upstream of the 
T7 promoter make better PCR transcription templates. A recent 
Biotechniques article on coupled in vitro transcritpion and 
translation mentioned that without any sequence upstream of the 
t7 promoter template, yields were dismal. My constructs contains only 
about 6 residues there, as indicated by the classic Uhlenbeck
article.

This arrangement gives me at best about 2 ug of RNA from a 50 ul
rxn containing 700 ng of template, which is sufficient for my 
purposes. Plasmid templates have given 5X more product routinely
in the lab. If I were to do it again, I would include a nice 
upstream region of 15 or so residues.

Jim
J. Graham



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