Primer Problem

Paul Cullen pcul at ozemail.com.au
Sat Feb 19 02:04:09 EST 2000


I regularly keep my primers in autoclaved double-glass distilled water and
have never had any difficulties.

> From: Warren Gallin <wgallin at gpu.srv.ualberta.ca>
> Organization: University of Alberta
> Reply-To: wgallin at gpu.srv.ualberta.ca
> Newsgroups: bionet.molbio.methds-reagnts
> Date: Fri, 11 Feb 2000 18:22:00 -0700
> Subject: Primer Problem
> 
> I have encountered a new problem and am at the end of my rope.
> I am getting big (both in terms of intensity and in terms of size)
> smears of DNA from PCR reactions.  It appears to be something odd with
> my primers, since a) I get the smear if there is no template present and
> b) increasing template decreases the amount and size of the smear DNA,
> indicating to me that the legitimate PCR product is in competition with
> this smear amplification.
> We have tried a variety of things, including increasing temperature
> and adding DMSO, glycerol and betaine; none have these have solved the
> problem.  It has happened with several different batches of primers,
> designed to recognize a number of different templates.
> I am considering the possibility that the problem may be that I am
> dissolving the primers in water, rather than TE, and that as a result I
> am getting some depurination that is making a small fraction of the
> primers relatively non-specific, causing the formation of a set of
> gradually concatenating primer products.
> So, my questions are:
> 1) has anyone else encountered this problem, and if so how did they
> fix it?
> 2) does anyone know whether dissolving primers in water (pH of the
> water is 5.6, who knows what the primers do to that) can cause this kind
> of problem or can depurinate a small fraction of the primer?
> 
> I would appreciate any and all suggestions.
> 
> Thanks,
> 
> 
> 





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