PCR ghost bands
oliver at molgen.biologie.uni-marburg.de
Fri Jun 24 08:46:50 EST 1994
In article <2uca3i$lp9 at apakabar.cc.columbia.edu> pcj1 at konichiwa.cc.columbia.edu (Pierre Jelenc) writes:
>From: pcj1 at konichiwa.cc.columbia.edu (Pierre Jelenc)
>Subject: PCR ghost bands
>Date: 23 Jun 1994 15:35:14 GMT
>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
you might have secondary structure in your template, which could lead to
deletions upon PCRing. If that is the reason you could prevent that by
adding ssb to the reactions.
For further information see: Q. Chou (1992), NAR, 20: 4371
Hope this helps.
Novell-Server Molekulargenetik Biologie Uni-Marburg Deutschland
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