Freeze-thawing of primers

Paul N Hengen pnh at
Wed Dec 4 12:53:40 EST 1996

David MacHugh (dmachugh at wrote:

> 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. 

Cool! But, why are you doing it?...just curious (my mummy wants to know) ;-)

> 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?

Yes, we've seen this before. I doubt that it's your DNA if you've seen it
work great and then less and less product is amplified from the same lot
of template over several weeks time. Check out this article:

author = "P. N. Hengen",
title = "Methods and reagents - Wayward {PCR} primers",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "1",
pages = "42-44",
month = "jan",
year = "1995"}

* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
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