Joseph C. Bagshaw
jbagshaw at wpi.edu
Sat Feb 12 08:05:44 EST 2000
Sounds like one of your components, most likely your polymerase, is
contaminated with stray DNA. Taq polymerase, and probably others, is
produced from a cloned gene expressed in E. coli, and the initial prep
contains plasmid DNA. If the vendor doesn't do a scrupulous job of
cleaning up the enzyme, some plasmid DNA is left. If theyr'e really
sloppy there might be scraps of chromosomal DNA as well. Try this: Make
up three identical reactions and set one aside without cycling. Add
template to one of the remaining two, then cycle these two and run all
three on a gel. If the smear is absent in the unincubated sample and
appears in the absence of added template, there is an unwelcome template
in the mix.
******************** HAVE GENES, WILL TRAVEL ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.
On Fri, 11 Feb 2000, Warren Gallin wrote:
> 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.
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