## Melting Temperature (Tm) CalculationSource code is freely downloable at biophp.org |

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:**

Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50
mM Na^{+}, and pH 7.0.