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