T_m for Oligos Calculator

The most sophisticated T_m calculations take into account the exact sequence and base stacking parameters, not just the base composition(1,2,3).

The equation used is:

$\text{T}_\text{m} = {\Delta\text{H} {\text{kcal}\over ^\circ\text{C}\: \times\: \text{Mol}} \over \Delta\text{S + R In ([primer] / 2)} } - 273.15\:^\circ\text{C}$

$\Delta$H is the enthalpy of base stacking interactions adjusted for helix initiation factors (3,4).

$\Delta$S is the entropy of base stacking adjusted for helix initiation factors (3,4) and for the contributions of salts to the entropy of the system (3).

R is the universal gas constant: $1.987\:\text{Cal / }^\circ\text{C} \times \text{Mol}$

Most melting temperature calculations do not take into account the effects of magnesium on helix stability. Therefore, most empirical guidelines used to design experiments will not apply when the magnesium effects are included. We have included the option to consider magnesium in the equation if it is desirable but have not included it in the default setting. Including magnesium will generally raise the theoretical melting temperature by about 5-8°C for oligonucleotides in a 1.5mM Mg²⁺ solution (5,6).

1. Rychlik, W. and Rhoads, R.E. (1989) Nucl. Acids Res. 17, 8543.
2. Borer P.N. et al. (1974) J. Mol. Biol. 86, 843.
3. SantaLucia, J. (1998) Proc. Nat. Acad. Sci. USA 95, 1460.
4. Allawi, H.T. and SantaLucia, J. Jr. (1997) Biochemistry 36, 10581.
5. von Ahsen N. et al. (1999) Clin. Chem. 45, 2094.
6. Nakano S. et al. (1999) Nucl. Acids Res. 27, 2957.