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PCR Annealing Estimate from Primer and Product Melting Points

Estimate a starting PCR annealing temperature with the Rychlik relationship and convert Celsius, Fahrenheit or kelvin measurements.

  • Formula & worked example
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Calculator inputs

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How to use this calculator

  1. Enter the known values in the units shown. Results update as you type.
  2. Where results are editable, change one to solve backwards. Lock a value to hold it fixed.
  3. Use the worked example to check the method. Reset restores the starting fields.

Private by default

Inputs and results stay in this browser tab. Bookify does not upload or store the values you enter.

Formula and method

A 60 °C primer melting point and 85 °C product melting point give a starting estimate of 62.6 °C.

Ta = 0.3 × Tm(primer) + 0.7 × Tm(product) − 14.9, with equation temperatures in °C.

Worked example

Enter these known values and leave the other values blank.

Primer melting temperature
60 °C
Target melting temperature
85 °C
Annealing temperature (Tₐ)
62.6 °C

Assumptions and limitations

  • Use the lower melting point of the primer pair and a product melting point determined under the intended assay conditions. The relationship is an empirical starting estimate; verify performance with a PCR temperature gradient.
  • The captured operating domain requires all three equation temperatures above 0 °C. This is a model limit, not absolute zero. Input unit conversions include temperature offsets.
  • The retained 50–65 °C note is a contextual band; enzyme chemistry, sequence, salt and assay conditions can require another temperature.

Common questions

How should I solve for another quantity?

Clear the unknown field and enter the independent measurements. A supported inverse preserves the same model; it does not add experimental evidence.

References

The calculation equations, inverse formulas, units, and input rules were imported from this source. Bookify provides the interface and equation solver.

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