HELP!!! PCR Smear!

Brian Foley brianf at med.uvm.edu
Tue Aug 2 15:08:37 EST 1994


Bengt Oxelman (bengt.oxelman at systbot.gu.se) wrote:
: I am sequncing rDNA from diverse green plants. For a lot of them, there
: have been few problems. Now I have some templates where the desired
: fragment seems to be impossible to amplify. All I get is a 'smear' of
: various lengths. My old, succesfully amplified templates works still
: though. My standard method for DNA extraction includes SDS/Mercaptoethanol,
: phenol/chloroform extraction and salt/ethanol precipitation. I have also
: used RNAse and CsCl sometimes, but it apparently didn't affect the outcome
: of the PCR reactions. 
:  ... Discussion of conditions deleted to save space here...

	Have you considered the fact that a SINGLE base mismatch will
prevent polymerization if it is the very 3' base that is mismatched?
TAQ polymerase will not extend from a primer in which the 3' end
is not perfectly paired.
	This can be "fixed" by using Pfu polymerase (or a mixture of
Pfu and TAQ) because the proofreading ability of the Pfu will chop
off the mispaired base and then extend.


--
********************************************************************
*  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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