Freeze-thawing of primers

David MacHugh dmachugh at mail.tcd.ie
Mon Dec 2 06:54:10 EST 1996


Hi,

I have been trying to amplify control region segments from ancient cattle
bones and have run into a problem.  The bones range from 1,000 year-old
samples from Viking Dublin to 12,000 year-old aurochs material. 
Initially, the PCR was working fairly well and I was getting enough
product to allow direct sequencing.  I have been amplifying two segments,
a 150 bp region and a 350 bp region - both of which worked.
However, in the last week or two the yield has dropped off dramatically
and a new primer-artifact has appeared of approximately 80-90bp.  This
artifact appears in all samples except the positive DNA control (ancient
samples, bone extraction control, normal extraction control and PCR
control).  Obviously, this artifact is reducing the yield of the target by
competing for primer.  I have a number of ideas as to why this may be
happening, but the most likely reason in my opinion may be due to
freeze-thawing degradation of the primers resulting in the appearance of
this primer artifact.  The PCRs were working perfectly when I first
received the primers (about 2 months ago).  Also there is no 3'
complimentarity between the oligos.

Has anyone had a similar experience, and if it is a recognised problem
with difficult or degraded template, should I store my primer stocks at 4
degrees C instead of -20 degrees C?

Thanks in advance,


David.

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*         (__)  David MacHugh PhD,   E-mail: dmachugh at mail.tcd.ie   *
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