Hi 'Netters,
We're having all sorts of complications with PCR from Chlamy genomic DNA
and hope that someone out there can help us out.
The basic problem is this: it seems as if most of the primers that we
design (against GC rich coding regions) work all too well by themselves -
control amplifications with only single primer generate lots of bands from
1 ug of genomic DNA (some of which are quite strong). Controls without
primer addition are usually clean. We are generally trying to use
degenerate primers (although the problem has happened with nondegenerate
ones too) and have tested a number of different enxymes including Taq, Pfu,
and Tfl. We have tried basic optimization steps (Mg++, additives) on the
reactions but cannot achieve specificity in reactions with foward and
reverse primer pairs. Raising the annealing temp (we generally work about
5degreesC below the lowest Tm) tends to eliminate all bands in a reaction -
unfortunately specific forward/reverse products do not appear.
Can anyone help us out? Is this a problem related to GC rich templates, or
are we missing something really obvious? I tried searching the newsgroup
and found others with PCR questions but few answers; if there is a good
response to my question I will post a summary. Thanks in advance for your
replies!
Karl Johnson
Haverford College
Karl Johnson
Assistant Professor of Biology
Haverford College
370 Lancaster Avenue
Haverford, PA 19041
tel 610-896-1306
fax 610-896-4963
kjohnson at haverford.eduhttp://www.haverford.edu/biology/
