Does Taq Stoffel fragment produce 3' A overhangs.

Michelle Gleeson michelle at MOLECULE.BIO.UTS.EDU.AU
Wed Aug 28 21:45:17 EST 1996



On Thu, 29 Aug 1996, Jamie wrote:

> We've been using Perkin-Elmer AmpliTaq Stoffel fragment for DNA
> fingerprinting and would like to clone some of the pcr products. We've
> been having troubles using Promega's pGEM-T kit and were just
> wondering if anyone knows if this enzyme produces 3' A overhangs as
> taq polymerase does. Also, what do people think of the pGEM-T kit, we
> seem to have got a few duds from them. Thanks alot.
>
> Jamie Nourse.
>
Hi,

I can't comment on the polymerase, but in our lab we've also found the
pGEMT kit to be quite variable.  They recently changed the protocol quite
a bit, and now recommend an overnight ligation at 4 degrees celsius,
S.O.C medium to resuscitate (with shaking), and to aliquot the ligase
buffer into small amounts on arrival, to avoid freeze thaw cycles.  When
I changed to the new protocol, the results improved markedly.  The
problems we used to have were, I think, mostly due to degrading ligase
buffer, as the kits seem to work fine the first couple of uses and then
stop.  I hope this helps, good luck

Michelle>




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