PCR-trouble w/t genomic DNA

Marcus Wernitz wernitz at landw.uni-halle.de
Mon Sep 7 08:22:27 EST 1998


Hi,

We are having trouble in our lab amplifying PCR-products out of genomic
DNA from a filamentous fungi.
We isolated genomic DNA with a plant kit by QIAGEN and it looked OK. We
also used an older preparation wich worked in the past (per
10microliters 50-100ng)
For the PCR we use degenerated primer-pairs for differetn genes we like
to clone. Quite a few times we produced bands on the gel with the
correct size. But trying to reproduce this result fails.
There is no problem amplifying fragments from plasmids with the same
primers, but it almost never works twice with genomic DNA.
We changed almost everything: New primers, new primer-dilutions,
diffrent amounts of magnesium, primers and polymerase. Different sources
of water. New buffer-solutions, new polymerase-vials, different
thermocyclers, varying annealing temperatures and times. Adding DMSO.

NOTHING

We use the taq and buffer by MWG, manufactured in Israel and pwo by
Boehringer Mannheim.

Can anybody see whatelse might be changed or what can be done to get
those once-seen-bands (OSB)?

Yours,

Marcus Wernitz
Uni Halle
Landw. Fak.
Inst. f. Phytopathologie
06108 Halle

Wernitz at landw.uni-halle.de



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