primer-dimers in pcr

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Nov 2 15:56:51 EST 1994


sl6dp at cc.usu.edu  wrote:

> Does anybody knows how to get rid of the nasty primner-dimers. My
> oligos have a lot of G-C. Something to do with the MgCl concentrations???

You can try to increase the annealing temp. to the minimum tolerated
by the  primer pair.  Reducing Mg has the same effect as increasing
annealing temp. and is convenient because you can try several Mg
conc.'s at once.  However, I don't recommend doing Mg titrations
because Mg alters several parameters of the experiment at once and
makes you vulnerable to a variety of secondary problems. 

If you can get the correct target band in your positive control, you
can probably ignore primer dimers.  But be careful to simulate the same
concentration/complexity in the positive control as in your target
template or else an inefficient ampl. might limp by on the positive
control but fail on the real thing.  
  
Otherwise, you're going to have to redesign the primers so they don't
prime on themselves or each other. It helps to make the 3' end the
least stable part of the primers. That is, don't put G+C clamps on 3'
ends unless something about your experiment absolutely requires it.

Hope this helps.
Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu



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