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Different Tm - (Jul/08/2005 )

When the primers have different Tm. How can I calculate the annealing temperature for the PCR? Should I take the average of the Tm for calculation?


Hi indoubt,

You have to use the lower annealing temperature. Because that is the temperature that that primer will anneal to the DNA if you take the average your it will be to high that primer won't anneal and no product will result.




scott is right
the lower Tm is the limiting temperature for annealing