PCR SMEARING

Deanne Bell dbell at qnis.net
Thu Nov 9 12:52:02 EST 2000


Hi Philip
My first reaction to your e-mail was that your annealing temperature during
PCR is too low.  So you may want to try a little experiment testing a range
of annealing temps (a range of 10 - 15 degrees, increasing by steps of 3
degrees).  If annealing temps are too high, the fragments will start
disappearing, if the temp is too low, you get a smear.
I am not sure what polymerase enzyme you are using but after setting-up
your reactions on ice maybe you could try a preliminary step of 2 min @
94*C at the beginning of your PCR.  This should not harm your enzyme but it
would ensure total denaturation.  Also PE Biosystems makes a polymerase
called Amplitaq gold that can be added to the master mix at RT and then
requires a prelim heating of 10 min @ 94*C before the cycling.  It is
probably more expensive than otehr polymerases, but it works consistently
everytime :-)
Also, did you run a gel (0.8 - 1.0% agarose) with just the DNA to check the
quality of the DNA you are starting out with? You should get a nice tight
band high up on the gel. If you get a smear here, your DNA is of poor
quality.
Another suggestion is to try optimizing DNA & MgCl2 concentrations.  Are
you running your fragments out on acrylamide or agarose? Is agarose at the
correct % for the fragment sizes you are looking at? In general, 1.5% -
2.0% should be used for fragments 300bp - 2000bp in size.

Hope this helps
Deanne Bell
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: dbell at qnis.net
phone: 559 453-3170
fax: 559 453-3088

----------
> From: "Philip O'Brien" <obrien at central.murdoch.edu.au>
> To: methods at hgmp.mrc.ac.uk
> Subject: PCR SMEARING
> Date: Wednesday, November 08, 2000 10:55 PM
> 
> We have been getting a lot of smearing in our PCR reactions.  Sometimes
we
> get a discreet band, but most of the time its a smear.  We get this with
> different primer pairs so it dosent't appear to be confined to one pair.
> The reactions are all set up on ice to minimize aberrant priming before
> thermalcycling.  I would be grateful if anyone could give me information
on
> how to eliminate the problem.
> 
> 
> Phil
> 
> Philip O'Brien
> Division of Science
> School of Biological Sciences & Biotechnology ,
> Murdoch University,
> Murdoch WA 6150,
> AUSTRALIA
> 
> Ph Nat     61-8-9360 2785
> Fax         61-8-9310 7084
> http://www.murdoch.edu.au/home.html
> 


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