PCR primer mismatches

Timo Hiltunen bltihi at uta.fi
Wed Nov 30 02:03:26 EST 1994


Are you sure that the size is smaller than expected ? It may only look
like that, because your size marker and PCR reaction sample may migrate
differentially due to different salt concentrations etc. 
In almost all my PCRs, for example, if I'm expecting a band of 410 bp, 
I seem to get a band that is 5-10 % shorter, but cloning and sequencing
confirms the product's right size.

Additionally, I have noticed that many size markers are not very accurate
and may have rather big differences when compared to other markers.

Timo Hiltunen
Univ. of Tampere, Finland

Dale R Shepard (dshepard at magnus.acs.ohio-state.edu) wrote:
: I am having a problem with an upstream primer which binds to a secondary site 
: on my template DNA.  In reactions with both upstream and downstream primers and
: template DNA, I get a band smaller than my expected 1.8 kb (this is the only 
: band).  I get the same band with only the upstream primer and the template DNA.
: I have tried three different primers and always have this occur (with different
: sizes pieces for each primer).  I am using Perkin Elmer AmpliTaq polymerase and
: I have used a couple of different reaction conditions.  My reactions have 
: contained 0.25 uM or 1 uM primers, 2.5 mM Mg chloride and 0.2 mM dNTPs.  My 
: temperature programs have been with a 45 C annealing temperature or a 55 C 
: annealing temperature during each amplification cycle, or with a combined 72 C 
: annealing/extension for each cycle.  All these conditions have given me the 
: same result.  My primers have been 19-, 24- and 27-mers.  Comparing the primer 
: sequences to the template DNA with the Fasta command on GCG, the binding site I
: want is about 87-95% similar to the template over the whole length of the 
: primer, while the secondary sites are about 90 % similar to a 7-10 bp overlap.
: Should I change my primers again? (I'm trying to introduce a BamHI site), use 
: an enhancer (like the one Stratagene sells), or change my reaction conditions?
: Any help would be appreciated.  Sorry for the length of the message, but I 
: didn't want to cause confusion.  Thanks in advance, I can post a summary of the
: responses.

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