Optimum temp for PCR primer annealing

mmg6 mmg6 at psu.edu
Fri Mar 24 01:31:33 EST 1995


In article <22MAR95.10855568.0089 at SEMOVM.SEMO.EDU>
Allen Gathman <C741SCB at SEMOVM.SEMO.EDU> writes:

> Does anyone have an algorithm for calculating the optimum temperature to
> use for annealing PCR primers, based on sequence and length?  I know
> that many commercial programs will do this, but we don't have one.  Of
> course, if anyone knows where I can get a shareware program that does
> primer design, I'd be interested in that too.


For primers 20 bases or fewer the Tm can be calculated by assigning 4
degrees C per G or C and 2 degrees for an A or T.  Use Tm minus five
degrees for your anealing temperature.  This simple calculation has
worked well in numerous PCR experiments in my lab.  Good luck

Cheers,

Michael



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