# 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. > Canonical page: https://elysiatools.com/en/tools/annealing-temperature-calculator - **Category:** Education - **Keywords:** annealing temperature, pcr ta calculator, tm minus 5, rychlik formula, primer annealing, pcr optimization, gradient pcr, primer tm, pcr protocol, primer design, pcr, molecular biology ## 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. ## Inputs - **Calculation Rule** (select): Simple rule uses the lower primer Tm; Rychlik also weighs the product Tm. - **Forward Primer Tm (°C)** (number): Melting temperature of the forward primer. - **Reverse Primer Tm (°C)** (number): Melting temperature of the reverse primer. - **Product Tm (°C, Rychlik)** (number): Amplicon melting temperature (typically 75–90°C for a 100–500 bp product); used by the Rychlik formula. - **Decimal Places** (number) ## 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 - Enter the forward primer Tm, reverse primer Tm, and product Tm. - Select the Simple rule or Rychlik formula calculation method. - The simple rule subtracts 5°C from the lower primer Tm; the Rychlik formula calculates 0.3 × primer Tm + 0.7 × product Tm − 14.9. - The 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. ## Frequently asked questions ### 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. ## Related tools - [Autosomal Dominant Pedigree Analyzer](https://elysiatools.com/en/tools/autosomal-dominant-pedigree): Compute AD offspring risk (Aa×aa 1:1, Aa×Aa 3:1, AA×any all affected), chi-square fit of observed children, and hallmark checks including male-to-male transmission that rules out X-linked dominant. Derived from Griffiths, Hartl & Clark 2007, StatPearls, OMIM. 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