Are these primer dimers?
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Wed Feb 12 03:01:42 EST 1997
In article <hopkinsc.120.000D3024 at lincoln.ac.nz>, hopkinsc at lincoln.ac.nz
(Hopkins, Charlotte) wrote:
> Hi all,
> We have a postgrad student who is starting to have problems with PCR that has
> got us confused. It is looking like she has two sets of primer dimers her
> gels. Both bands occur under 100bp in length,and it is clear that they are
> two distinct bands (I would suspect about 50 and 90 bp - our ladder
> below 100bp so I can't be more specific). We suspected the top one to be
> contamination, as it also occurs in the negative, so tried all new chemicals,
> but they are still there. It doesn't always occur in every tube either
> (perhaps 3 out of the 5) and there is no correlation with individuals or
> species. We have tried a different DNA region (i.e completely different
> primers) and they still occur.
> This morning she went to rerun samples that were 5 days old and her expected
> band had completely disappeared but she still had these two blobs at the
> bottom. We then thought DNAses could be contaminating the samples while they
> are in the fridge, however she has just taken sample straight out of the PCR
> machine and have run them on a gel and the bands are present. Could it be
> DNAses or should the PCR destroy them? If this problem is being caused by
> DNAses why doesn't it happen in all tubes as a master mix of all but DNA is
> made up each time?
> Has anyone experience this or can explain what is likely to be causing it?
> Many thanks
> Charlotte Cameron email: hopkinsc at lincoln.ac.nz
> Lincoln University
> New Zealand
Please clarify what polymerase you use and what your conditions are (how
much primer, nucleotides, template, cycles etc.). Otherwise it will not be
possible to anwser your particular question.
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
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