Hot Start PCR failures

James Prescott prescj at dianon.com
Thu Dec 19 18:20:32 EST 2002


I am trying to set up hot start PCR to amplify genomic DNA isolated
formalin-fixed tissue.

The wax barrier method would work best, but trying home made wax
pellets of two different waxes yeilds highly variable results, with
between a third and half of the reactions failing using control human
genomic DNA. Controls consisting of mixing the upper and lower
reactions work great, and all reactions (including controls) are from
the same master mix.

We tried a sample of magensium-wax beads again with highly variable
results (controls worked great).

Why wax barrier? I am using Expand Hi-Fi for the high fidelity (a must
for a mutation detection assay) and the lower royalties. Wax barrier
keeps me from having to re-charaterize the entire assay since I can
use the same enzyme mix.

Why hot start? Because trying to get good length (600bp+) DNA from
formalin fixed tissue sections sucks and normal PCR from this type of
template gives very intense artifacts with a corresponding loss of
signal.

What is people's experience with different waxes, different wax
volumes, etc., or has everyone just switched to heat activated Taqs?
Are there any suggestions for increasing the reproducibility of the
reactions? Does anyone have any evidence of wax inhibiting the PCR or
is it most likely a mixing problem?

TIA,
James



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