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)

ASSUMPTIONS:
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.