In vitro transcription, Ambion's secret?

Eric A. First EFirst at lsumc.edu
Thu Jul 6 15:15:53 EST 1995


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)





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