PCR ghost bands

Robert Preston rapr at MED.PITT.EDU
Thu Jun 23 17:46:46 EST 1994


> From: pcj1 at konichiwa.cc.columbia.edu (Pierre Jelenc)
> Subject: PCR ghost bands
> 
> 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
> 
Pierre, how many years lab experience do you have? Where do you get
your primers? Have you ever checked the primers by HPLC or whatever?
How many different primer sets has this happened with, and what were
the sizes of the primers involved.  Etc.  This kind of mystery probably
needs more data to solve.

Rob Preston
rapr at med.pitt.edu




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