Melting Temperature (Tm) Calculation

Primer (6-50 bases):
Basic Tm
Degenerated nucleotides are allowed
Base-Stacking Tm
Degenerated nucleotides are NOT allowed
Primer concentration: nM
Salt concentration: mM
Mg2+ concentration: mM
Source code is freely downloable at biophp.org

Basic Melting Temperature (Tm) Calculations

Two standard approximation calculations are used.
  • 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)

When degenerated nucleotides are included in the primer sequence (Y,R,W,S,K,M,D,V,H,B or N), those nucleotides will be internally substituted prior to minimum and maximum Tm calculation. 

    Example:
    Primer sequence:                       CTCTRYCTWSCTCTCT
    Sequence for minimum Tm calculation:   CTCTATCTAGCTCTCT
    Sequence for maximum Tm calculation:   CTCTGCCTAGCTCTCT

ASSUMPTIONS:

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