PCR specificity

Hayward-Lester heall at ttacs1.ttu.edu
Fri Apr 15 12:09:00 EST 1994

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

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