PCR problems

Brain Foley brianf at med.uvm.edu
Wed Nov 3 18:07:43 EST 1993


farleyp at HAL.HAHNEMANN.EDU wrote:
: Hello Netters!

: We have been experiencing problems amplifying certain templates.  When the
: agarose gel is run, we see a large smear of DNA over a broad molecular weight
: range.  We have tried different concentrations of MgCl2, template, primer, and
: even different PCR temperature profiles.  Does anyone know what causes this and
: how we might get better results?  Thank you in advance for any help that you
: can offer.

: Yang Kang &
: Patrick Farley
: e-mail address YANGK at HAL.Hahnemann.edu

	There are many things that can cause a smear.  That is what makes
trouble-shooting PCR so much fun.

	If the smear is larger than the size of the predicted product, you
may be "overdoing it".  I have found that any of the following can result
in such a smear:  Too much template, too much polymerase, too many cycles.
To trouble-shoot this, take out a small sample 1/2 and 3/4 of the way to
the last cycle.  i.e. if you are running 20 cycles, take a sample at 10
and 15 cycles.  You might see a nice band at 10 cycles, a bit more at 15,
and then just a smear at 20 cycles.
	If the smear is smaller than the expected size, you might just be
looking at primer dimers and other artefacts.  Run one tube with no
polymerase added and another one with no template DNA added.  Make sure
that the smear only occurs when BOTH template and polymerase are present. 
If not, then the smear is not template-dirived PCR product.
	
	That is the only advice I can give you unless you get specific
about things:
	What is your template?  Genomic DNA, plasmid, cDNA...
        How many nanograms of template are in your reaction?
        How many cycles?
        What are your primer sequences?
        How many picomoles of each primer do you add?
        What is the predicted size of the PCR product?
        Do you use mineral oil overlay?
        Do you do 50 or 100 microliter reactions?
        What "brand" of polymerase are you using?
        etc...
        What positive and negative controls have you run?
        What were the results of those control reactions?

 --
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



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