Troubles with Pfu polymerase

[Yves hatzfeld]

Hi Netters!

I have troubles with amplifying a 1.5 kb fragment with pfu polymerase
(stratagene). I tried first the "basics" conditions (as they write in
the documents supplied with the enzyme), but I had nothing at the end,
even in the positive control. I then tried to modify the amount of
template (5 times more), of dNTP, of oligos or of pfu, or I lovered the
annealing temperature from 40¡C to 30¡C (!) without any success. I even
tried a control experiment using a couple of oligos, a template and PCR
conditions  which worked well previously with pfu, without success,
despite the fact that my pfu is fresh new. 

When I do the same experiments using Taq instead, I haven't any problem
and I obtain the expected products ...

So why the Hell it does not work? I need to use pfu because I have to
make a protein fusion with the PCR product, and I don't want to have to
much chances to have some mutation (ie I don't want to sequence my
gene, if possible).

Any idea would be very wellcome, before I encounter a nervous
breakdown...

Thanks in advance, and Happy New Year!

Yves.