How this calculator works
The empirical relation weights the less stable primer-template melting temperature and the amplified product melting temperature. Its coefficients apply to Celsius temperatures; input units are converted before substitution. Use melting temperatures calculated for your actual salt and primer conditions. A temperature gradient remains useful when transferring protocols.
Formula
Ta (C) = 0.3 × Tm,primer (C) + 0.7 × Tm,product (C) - 14.9. Reverse modes rearrange this linear relation for either melting temperature.
Worked example
A primer melting at 60 C and a product melting at 80 C give 18 + 56 - 14.9 = 59.1 C. With primer 50 C and product 70 C, the estimate is 49.1 C.
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Frequently asked questions
Which primer melting temperature should I use?
Use the less stable primer-template pair, normally the lower of the two appropriate melting-temperature estimates.
Does this replace polymerase instructions?
No. Buffer chemistry and specialized polymerases may require another annealing rule. Follow the manufacturer's protocol and verify the product.
Can I enter Fahrenheit?
Yes. The calculator converts Fahrenheit or kelvin to Celsius before applying the empirical coefficients and converts the result for display.