Home-brewed T7 RNA polymerase vs Commercial T7 RNA pol
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.
More information about the Methods