Help: high mutation rate during RT-PCR

Robert Hartley rh at mblab.gla.ac.uk
Tue Aug 15 07:35:36 EST 2000


In article
<B7868D2D2540D311BED900105AD9DBA957E41B at n4s03451.grunenthal.com>,
Clemens.Gillen at grunenthal.de ("Gillen, Dr. Clemens") wrote:

Hello Clemens.
> Hi everybody,
> we are using the Advantage polymerase to amplify genes via RT-PCR and =
> we
> face the problems of various mutations. Do you know of certain =
> experimental
> conditions or better polymerases to overcome this problem?

I think that advantage is a 1/50 ratio of a bit of proofreading enzyme and
normal Taq with a bit of Taq start Ab. I know that they use Advantage in
their ddRT-PCR Delta kit and you can sometimes get a spurious banding
pattern. (don't know the reason but this may be it)

You could use PFU from Stratagene. I beleive that is more accurate WRT
mutation rate. It is a bit pricey but so is a failed reasction or over
mutation. :-) 

I could dig out a reference for you WRT enzyme mutation rates but I am in
the middle of transferring from Endnote to !Citation and then BibTex for a
database manager. 

Give me a private e-mail if you have no sucess here and I'll have a hunt
at the weekend.

Cheers
Bob; Cloudy but WARM Scotland.






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