PCR specificity

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Fri Apr 15 14:54:59 EST 1994


A smear of the type that you describe is not unusual when the
amplification conditions are suboptimal.  I recommend that you optimize
your reaction conditions for Mg2+ concentration (I usually vary from
1.5-3.0mM), annealling temperature (trying going up to increase the
stringency), and Taq concentration for starters.  When some of these
factors are optimized, the smear usually goes away.

> Hi!
> I am trying to amplify product from mouse total RNA using RTPCR, and
> primers designed for the rat sequence.  This gene is highly conserved
> between species, in fact I can amplify from human total RNA.  The
> sequence for mouse is unavailable, so I cannot design mouse-specific
> primers.  I am getting a smear of high molecular weight material (most
> of it stays in the well) when the products from a reaction run using a
> moderately stringent annealing temprature are run out on a gel.  Does
> this indicate that one primer is annealing under these conditions?  I
> have tried lowering the temperature a few degrees, and this does not
> improve the results.  Does anyone have any advice they could offer?
> Thanks in advance
> Amanda  
> 

==============================================================================
James F. George, Ph.D.              "Back off man, I'm a scientist"
Department of Surgery                --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
===============================================================================



More information about the Methods mailing list