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.
Melting temperature (Tm) = 4 * Number of G or C + 2 * Number of A or T (°C)
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.
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(1M NaCl) + 11.7 * log10([Na+]) (°C)
(Jhon SantaLucia, Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998))