Annealing Temperature Calculator

OptiBind has been created to calculate the optimal annealing temperature (Ta) of a pair of primers based on the nearest neighbour modelling for their respective melting temperatures (Tm).

The nearest neighbour model considers the entropy and enthalpy of nucleotides based upon their specific arrangement in the DNA sequence. These values can then be adjusted for salt and primer concentrations to output a melting temperature. Information about the sequence length and G-C content are also provided.

Annealing Temperature

The formulas used for calculating the optimal annealing temperature is:

Optimal Annealing Temperature (Ta) = 0.3 x (Tm of primer) + 0.7 x (Tm of product) – 14.9

Melting Temperature

The formulas used for calculating the melting temperature based on a DNA sequence is:

Melting Temperature (Tm) = (1000 x ΔH) / (ΔS + R ln (DNA /2)) - 273.15

Annealing Temperature

The optimal annealing temperature (ta) is the temperature at which the primers and the target DNA molecule are most likely to bind and form a stable hybridization. The optimal annealing temperature is calculated using the following formula: ta = 0.3 x (Tm of primer) + 0.7 x (Tm of product) – 14.9, where...

Tm of primer is the melting temperature of the primer
Tm of product is the melting temperature of the target DNA molecule
"0.3" represents the fraction of the primer's melting temperature that contributes to the optimal annealing temperature
"0.7" represents the fraction of the target DNA molecule's melting temperature that contributes to the optimal annealing temperature
"-14.9" is a constant value that is used in the formula to calculate the optimal annealing temperature

It is recommended the difference in Tm between primers should be less than 5°C AND the annealing temperature should not exceed 72°C

Notes (will vary depending on experimental setup):

Tm of product will vary depending on your PCR experiment design
Primers should aim for a minimum GC content for 40%
Primers should aim for a minimum length of 7 nucleotides
Primers should avoid long repeat region

Melting Temperature

The melting temperature (Tm) is a measure of the temperature at which the double-stranded DNA structure of a molecule begins to denature and separate into single strands. The Tm is calculated using the following formula: Tm = (1000 x ΔH) / (ΔS + R ln (DNA /2)) - 273.15, where...

ΔH is the enthalpy of melting
ΔS is the entropy of melting
R is the gas constant
DNA is the concentration of the DNA molecule
"1000" is a conversion factor that converts the units of ΔH from calories per mol to joules per mol
"ln" is the natural logarithm function, which is used to calculate the logarithm of the DNA concentration value
"2" is a constant value that is used in the formula to calculate the Tm
"-273.15" is a conversion factor that converts the units of temperature from degrees Celsius to degrees Kelvin.

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