PCR amplifying vertebrate D-loop: multiple bands

[Andrew McArthur]

This from a colleague of mine.  Please reply to my email address.

    Andrew

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HELP.  I'm a new grad student and I'm trying to amplify a 1 kb region
of the D-loop (mtDNA) in some carnivores (bears, martens, weasels)
using the Kocher et al. (1989) universal primers (L 15926, L16007,
H00651). I consistently get two bands of similar molecular
weight (near 1.3 kb), in all amplifications.  I have tried diluting
my template, increasing annealing temperatures (> 60 degrees),
different magnesium concentrations (25 mM, 50mM, 100mM) all to no avail.
I tried nested PCR but different reactions tended to preferentially
amplify different bands.  Has anyone else got two products using
these primers under highly stringent conditions?  If not, I will be
forced to sequence both to find out which is my target.  If anyone out
there can offer me some advice it would be greatly appreciated.