In vitro transcription, Ambion's secret?

[Michael Morales]

: >Promega makes a similar high yield transcription kit which they
: >described in a recent issue of Promega Notes.  The article was very
: >complete including buffer recipes and final concentrations of all
: >the other components.  When I copied the protocol, it worked
: >fabulously.  I'm sorry I don't have the issue number right now
: >because I'm at home on holidays.  If you can't find it, e-mail me
: >next week when I'm back in the lab and I'll send you the details.
: >Jill

: Are you sure that this is a 'Mega' kit or just the recipes for normal in vitro 
: transcription reactions for those polymerases (T3,T7,SP6)?  Epicentre Tech. 
: also makes a kit similar to Ambion's called Ampliscribe and it works well 
: also, but I don't know the secrets.  I would like to know though, please reply 
: to me if you figure it out!

I get 80-90% yields (NTPs incorporated) using the method of Pokrovskaya &
Gurevich.  They add higher NTP concentrations, more enzyme, HEPES buffer, 
and pyrophosphatase.  It also works for capped RNA.  E-mail me if you're
interested.  Oh yea, the full reference is:
Pokrovskaya,ID & Gurevich,VV (1994) Analytical Biochemistry 220:420-423.

Good luck,
Mike Morales
mmorales at acpub.duke.edu