Home-brewed T7 RNA polymerase vs Commercial T7 RNA pol

the End jgraham at bronze.ucs.indiana.edu
Sun Nov 21 14:54:18 EST 1993



>PS. I hope this doesn't violate any patent.

Nonsense. "Owning" enzymes is only a recent proposal.

We also routinely purifiy our own T7 polymerase. I will find out the 
details and get back to you.

Lately I have been going with NEB T7 at 3ul/100ul rxn with 3mM NTP's
and Hepes-KOH. Using the conditions as described in the 
Gurevich et al. (Analytical Biochem. 195, 207-213. 1991) and adding 3 G's to
the intial 5' end of the transcript, I  am getting about 3-10 ug of gel 
purified RNA from PCR generated 300 bp templates. I would expect a plasmid 
template to give higher levels if it were practical, from previous preps
done by others here. 

Milligram quantities from a single reaction seems rather far off. However 
the Ambion procedure uses 7 mM NTPs. Perhaps a corresponding higher 
(>10 mM) Hepes would be appropriate.

Jim
J. Graham



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