In vitro transcription, Ambion's secret?

Jillian Johnston johnstoj at acs2.acs.ucalgary.ca
Mon Jul 10 16:12:27 EST 1995


jim hartley (jhartley at access2.digex.net) wrote:
: Have you tried pyrophatase?

: Jim Hartley

: On 6 Jul 1995, Eric A. First wrote:

: > Hi netters,
: >         We're trying to set up large scale in vitro transcription reactions
: > to synthesize tRNA. We've used Ambion's T7-MEGAshortscript kit with good
: > results.  We've also used a homemade kit with less than impressive results.
: > Swapping various parts between the kits tells us that the main difference
: > is in the enzyme mix used by Ambion (our homemade enzyme mix contains T7
: > RNA polymerase, RNasin, and acetylated BSA).  Does anyone know what Ambion
: > puts in their enzyme mix that makes it work so well (i.e. is there a
: > literature reference)?  We've tried adding as much as 50 units of T7 RNA
: > polymerase per 20 ul reaction and still get a 10-fold lower yield than with
: > the Ambion enzyme mix.  Any ideas or suggestions on how we can increase our
: > yield?  Thanks.
: > 
: > Eric First (efirst at lsumc.edu)
: > 
: > 
: > 
: > 
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



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