HELP! re-amplification problem

bo bo at morgat.udg.es
Wed Mar 7 11:40:25 EST 2001


Hi,

I need some help from an expert!

I designed a pair of degenerate primers directed to a specific gene of a
green sulfur bacterium. After amplification we could detect several bands in
the gel that match to our target gene according to its expected size. In
order to obtain a large amount of target, we planned to re-amplify it using
the same degenerate primer pair used in the first round. Accordingly, we cut
the suspected band and we washed the DNA with a standard kit. Afterwards, we
checked its integrity and concentration and we use it as template for a
second PCR. In this case, however, we couldn't see any band in the gel. We
repeated the PCR at low temperature (44 °C) to allow primer annealing but it
didn't work. We repeated all the process several times modifying different
PCR parameters (i.e. amount of template, nº of cycles, etc.) but all the
tests failed at the same point, that is, the re-amplification. However, the
first amplification gave us the suspected product.

Does anybody have any suggestion? All ideas will be welcome!
Thank you very much in advance!!



Carlos Borrego

_____________________________________________________

Microbiology Section, Institute of Aquatic Ecology
University of Girona
Campus de Montilivi, E-17071, Girona (Spain)
Phone:  +34 972 418261
FAX:     +34 972 418150
_____________________________________________________







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