How to Use the Primer Tm Calculator

Single Primer: Paste a sequence in the box. Adjust monovalent salt (Na⁺) or primer concentrations if they differ from standard standard PCR buffer settings. Wallace rule (optimal for <14 bp) and Nearest Neighbor thermodynamics (>14 bp) are outputted instantly.

Primer Pair: Input forward and reverse primer sequences side-by-side. The tool will check the melting temperatures, the Tm difference (ΔTm), and recommend an optimal annealing temperature. If the ΔTm is greater than 5°C, a warning badge will be triggered.

Batch Mode: Analyze up to 20 primers at once. Simply write one sequence per line, optionally prefixing the line with a label and a comma (e.g. Exon1_Fwd, ATGCGATC...).

Tm Calculation Methods

// Wallace Rule (short primers ≤14 bp):
Tm = 2°C × (A + T) + 4°C × (G + C)

// Nearest Neighbor (SantaLucia 1998, primers >14 bp):
Tm = ΔH / (ΔS + R × ln(Ct / 4)) - 273.15 + 16.6 × log10([Na+])
Where R = 1.9872 cal/(mol·K), Ct = primer concentration, [Na+] = sodium concentration. Includes initiation and terminal adjustments.

Primer Tm Calculator

Analysis Results

How to Use the Primer Tm Calculator

Tm Calculation Methods

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