How to use the Tm calculator

The Tm calculator is divided in two sections: Tm calculation and primer dilution. The Tm calculation section gives the Tm for a primer, counts the number of different bases and GC content, and determines the molecular weight and extinction coefficient. The primer dilution section gives instructions on how to make a stock solution, and how to dilute the primer to a desired final concentration.

Tm calculation

Type in or paste your sequence to the first field. Fill in the PCR conditions to the fields next to it (in case you need to change the default values). Tm values and other data are automatically generated.

The application is designed to calculate Tm's according to three different methods. This is mainly to illustrate that the results can vary significantly between different methods. The modified Breslauer's thermodynamics¹ is the method Finnzymes has used for optimizing PCR protocols , and it is recommended to be used when setting up reactions with Finnzymes' DNA polymerases.

Note that it is seldom optimal to use the Tm value directly as an annealing temperature in PCR. To determine which annealing temperature to use with different Finnzymes' DNA polymerases and kits, please read the instructions below.

Important instructions on annealing temperature

Primer dilution

Amount of primer
Fill in the OD₂₆₀ value for your primer, or the µg or nmol amount of the primer. The three fields are interconnected, meaning that changing one value will change the other two accordingly.

Preparing stock solution
Fill in the concentration you want your stock solution to have. The application automatically calculates the volume of H₂O or buffer needed to dissolve the entire amount of primer to the desired concentration.

Dilution
Fill in how much of your final dilution you want to make, and the desired final concentration. The application calculates the required volumes of stock solution and H₂O/buffer.

*Breslauer et al., Proc. Nat. Acad. Sci. 83, 3746-50 (1986)