PCR primer melting temperature

R. Mark Adams ma690188 at mbir.bcm.tmc.edu
Wed Feb 26 12:37:26 EST 1992




Here is the promised summary of the replies I got regarding PCR primer melting
temperature.  Thanks to all the folks that replied!  Some references are
appended at the end.  Note that there is not yet an adequate theoretical
underpinning for the calculation of oligonucleotide Tm when mismatches are
contained.  An empirical estimate of that value can be obtained by using the
Tm - 1.2*(%mismatch) correction to the Tm obtained by the described methods. 
This will probably be an underestimate for primers in which the mismatches
are clustered.  (see the BioTechniques reference, below.) The best way to 
calculate these values, however, is empirical.  For most purposes, PCR 
included, the calculations provided below are probably satisfactory.


Several formulas:


Tm = 81.5 + 16.6log[Na+] + 0.41(%G+C) - 600/N

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Where:
  Tm      = Temperature of melting
  (%G+C)  = Percent G and C composition
  N       = Length of Oligo in nucleotides.



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