Oligo melting temperature and PCR annealing temperature

Melting temperature is the temperature at which 50% of the oligo and its perfect complement are in duplex.

PCR annealing temperature a few degree (4-6) lower than the melting temperature is usually used to increase the probability of primer binding.

To calculate melting temperature or for more information, please see BioToolKit or Primo Online.

Simple rule

Melting temperature (Tm) = 4 * Number of G or C + 2 * Number of A or T (C)

Nearest Neighbor

The Nearest N predicts melting temperature using the "Nearest Neighbor" model (Jhon SantaLucia, Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998)), which is more accurate.

Alternative model

Calculations using the following formula

Tm = 64.9 + 41 * (Number of G & C - 16.4)/oilgo length - 500/oligo length (C)

are usually in agreement (± 3 C) with the results using the Nearest Neighbor model.

Correction for salt

Tm([Na+]) = Tm(1M NaCl) + 11.7 * log10([Na+]) (C)

(Jhon SantaLucia, Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998))