In vitro transcription, Ambion's secret?

zinc 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:
>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,

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.

ciao,

- -pjf

- -- 
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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