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What formula is used to calculate Melting Temperature(Tm)?

Two standard approximation calculations are used for Melting Temperature (Tm) Calculations:

  1. For sequences less than 14 nucleotides the formula is:
    Tm= (wA+xT) * 2 + (yG+zC) * 4

    where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

  2.  For sequences longer than 13 nucleotides, the equation used is:
    Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

Equations above assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0.


Please click the following link for an online Melting Temperature (Tm) calculator.