In vitro transcription, Ambion's secret?
zinc at zifi.genetics.utah.edu
Sun Jul 9 15:09:37 EST 1995
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In article <9507062010.AA20079 at unixsrv1.lsumc.edu>,
Eric A. First <EFirst at lsumc.edu> wrote:
> 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
try adding pyrophosphatase to the rxn. i believe this is one of the
*secret* ingredients. this enables the rxn to continue for a longer
period of time. this is also used with many sequencing kits
(sequenase) to read more seq.
patrick finerty = zinc at zifi.genetics.utah.edu = pfinerty at nyx.cs.du.edu
U of Utah biochem grad student in the Bass lab - zinc fingers + dsRNA!
** FINGER ME for my pgp public key ** CRYPTO FOR THE MASSES!
zifi is a 486 DX4-100 running LINUX 1.2.10, send me all of your RAM now!
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