PCR ghost bands
pcj1 at konichiwa.cc.columbia.edu
Thu Jun 23 10:35:14 EST 1994
Very often (3/4 of the time), I see a PCR product shorter than the correct
one. This extra band is sometimes much stronger than the correct one, and
the difference in size between correct and incorrect is pretty much random
from primer pair to primer pair. I.e. it is not a fixed number of
nucleotides, nor is it proportional to the size of the correct fragment.
This extra band is _always_ shorter, typically by 10-30 nucleotides, and
it is absolutely reproducible with a given primer pair, regardless of PCR
conditions (Mg concentration, annealing temperature, elongation time,
number of cycles between 20 and 40, etc). It is readily visible in
denaturing acrylamide gels, and seems to be there in concentrated agarose
gels as well, although the lower resolution in the latter makes it
difficult to be sure.
It does not appear to be a gel artifact, because I have run side by side
reactions with and without extra bands, nor to be simply due to the
reaction conditions, because I run several reactions from the same premix
of reagents, added to the template and primers that have been premelted
and annealed under wax.
I am at a complete loss.
Pierre Jelenc pcj1 at columbia.edu
Columbia University, New York
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