Tm Calculator

This tool calculates the T_m of primers and estimates an appropriate annealing temperature when using different DNA polymerases. How to use this calculator

Quickly find the right annealing temperature for Platinum SuperFi DNA polymerase (also works for SuperScript IV One-Step RT-PCR Kit), Phusion and Phire DNA polymerases.

Important note: If the PCR primer contains desired mismatches, e.g., for creating a mutation or a restriction site, make sure to calculate the T_m only for the correctly matched sequence

The T _m calculator is not required for Platinum II Taq DNA Polymerase, Platinum SuperFi II DNA Polymerase, and Platinum Direct PCR Universal Master Mix, and Phusion Plus DNA Polymerase due to their buffers specially formulated for a universal annealing temperature of 60°C for primers.

1. Select your DNA polymerase

Platinum SuperFi DNA polymerase
(Also select this option if using the SuperScript IV One-Step RT-PCR Kit)
Phusion or Phire DNA polymerase
DreamTaq DNA polymerase or other Taq-based DNA polymerase

2. Select input method

Single pair
Batch

3. Type or paste your sequence

Primer#1: 5′-

Primer#2: 5′-

4. PCR conditions

Primer conc. µM

Results

Export table data into Excel

ID #1	Sequence #1	Molecular weight g/mol	Extinction coefficient l/(mol˙cm)	Tm °C	ID #2	Sequence #2	Molecular weight g/mol	Extinction coefficient l/(mol˙cm)	Tm °C	Annealing Temperature °C

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How to use the T_m calculator

The calculator calculates recommended T_m (melting temperature) of primers and PCR annealing temperature based on the primer pair sequence, primer concentration, and DNA polymerase used in PCR. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient.

The modified Allawi & SantaLucia's thermodynamics method [1] is used for T_m and annealing temperature calculation of reactions with Platinum SuperFi, Phusion and Phire DNA Polymerases. The parameters were adjusted on a set of primers seeking to maximize specificity and retain high yields.

To use this calculator select your DNA polymerase, type in or paste your primer sequences, and provide your final primer concentration. T_m values, annealing temperature, and other data are automatically generated.

If necessary, use a temperature gradient to further optimize and empirically determine the ideal annealing temperature for each template-primer pair combination. The annealing temperature gradient should start with temperature 6–10 °C lower than annealing temperature generated by the calculator and increased up to the extension temperature (two-step PCR).