Primer Problem

khalil khalilha at sympatico.ca
Sat Feb 12 20:06:12 EST 2000


Hi,

I always use water to resuspend my primers and never had any problems.
The only explanation i could have is primers annealing to each others and
could cause the smearing.
Increasing the concentration of template will decrease the smearing by
competition.
You should check the following :
        1) Your primers doesn't have complementary sequences between them
        2) Does one of your primers possess complementary sequences
                        This would cause hairpin but also large annealings

                ex:     5'  AAATTTxxxxxxxxxxxxxxxxxAAATTT  3'

5'  AAATTTxxxxxxxAAATTT  3'
                            3'  TTTAAAxxxxxxxTTTAAA  5'
                                                         5'
AAATTTxxxxxxxxAAATTT 3'


"Joseph C. Bagshaw" <jbagshaw at wpi.edu> wrote in message
news:Pine.OSF.4.21.0002120756020.16726-100000 at grover.WPI.EDU...
> 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.
> >
> > Thanks,
> >
> >
> >
>






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