PCR Annealing Temperature Calculator (Ta = Tm − 5°C)
PCR Ta by the simple rule (lower primer Tm − 5°C) or the Rychlik 1990 formula (0.3·Tm_primer + 0.7·Tm_product − 14.9), with a ±3°C gradient window and primer-Tm mismatch warning. Derived from Rychlik 1990, Innis & Gelfand 1990, Lorenz 2012. Lab-planning use only.
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Tool usage guide
Learn when to use this tool, what it supports, and how real users apply it.
Key facts
Category
Education
Input types
select, number
Output type
json
Sample coverage
4
API ready
Yes
Overview
The PCR Annealing Temperature Calculator estimates Ta from forward and reverse primer Tm values using either the simple lower-Tm-minus-5°C rule or the Rychlik 1990 formula, which also uses product Tm. It returns the selected Ta, a ±3°C gradient range, and a warning when primer Tm values differ too much. Use the results for lab planning only.
When to use
Choose an initial PCR annealing temperature from a primer pair's forward and reverse Tm values.
Plan a gradient PCR using the calculated Ta and its ±3°C testing window.
Compare the simple Tm − 5°C rule with the Rychlik 1990 formula when product Tm is available.
How it works
1Enter the forward primer Tm, reverse primer Tm, and product Tm.
2Select the Simple rule or Rychlik formula calculation method.
3The simple rule subtracts 5°C from the lower primer Tm; the Rychlik formula calculates 0.3 × primer Tm + 0.7 × product Tm − 14.9.
4The calculator reports the selected Ta, a range extending 3°C below and above it, and a primer-Tm mismatch warning when applicable.
Use cases
Set an initial annealing temperature for a new primer pair.
Define a gradient PCR range when optimizing specificity and amplification.
Cross-check a simple Ta estimate against the Rychlik formula for a known product Tm.
Examples
1. Standard primer pair using the simple rule
PCR researcher
Background
A primer pair has forward Tm 62°C and reverse Tm 60°C, with an estimated product Tm of 85°C.
Problem
Choose an initial annealing temperature and a practical gradient range.
How to use
Select Simple rule, enter forwardTm 62, reverseTm 60, productTm 85, and set Decimal Places to 1.
The calculator returns Ta = 55°C with a 52–58°C gradient range. The 2°C primer-Tm difference does not trigger a mismatch warning, and the Rychlik cross-check is 62.6°C.
2. Rychlik formula for a higher-temperature comparison
Molecular biology student
Background
The same primer pair has Tm values of 62°C and 60°C, and the product Tm is 85°C.
Problem
Estimate Ta with the Rychlik 1990 formula and compare it with the simple rule.
How to use
Select Rychlik formula, enter forwardTm 62, reverseTm 60, productTm 85, and set Decimal Places to 1.
FAQ
Which primer Tm does the simple rule use?
It uses the lower Tm of the forward and reverse primers, then subtracts 5°C.
What is the Rychlik formula?
It is Ta = 0.3 × primer Tm + 0.7 × product Tm − 14.9.
What does the gradient range mean?
It is a suggested testing window extending 3°C below and above the calculated Ta.
Why is a primer-Tm mismatch warning shown?
The warning indicates that the forward and reverse primer Tm values differ beyond the recommended range.
Is this calculator a complete PCR protocol?
No. It provides temperature calculations for lab planning and does not replace assay-specific optimization.