PCR Primer Tm Calculator
Paste your DNA primer sequences and see their melting temperature calculated instantly. Automatically checks for hairpin loops and structures that could cause your experiment to fail. Nothing leaves your browser.
Forward Primer
Learn more: PCR primer Tm and thermodynamic calculations
Nearest-neighbor method vs Wallace rule - why precision matters
The Wallace rule (Tm ≈ 2(A+T) + 4(G+C)) is simple and fast, but treats all base pairs equally. Nearest-neighbor (SantaLucia 1998) sums enthalpy and entropy contributions of each adjacent base pair, accounting for stacking interactions. For primers longer than 18bp or with unusual GC content, nearest-neighbor is substantially more accurate.
Salt concentration and DMSO - how buffer composition changes Tm
Primer Tm is calculated in reference salt (1M NaCl). Your PCR buffer is typically 50mM KCl and 1.5-3.5mM MgCl2. Higher salt increases Tm; the calculator corrects for this. DMSO, added for GC-rich regions, decreases Tm. Both corrections are essential for getting annealing temperature right.
Hairpin and dimer detection - catching self-binding problems
A hairpin forms when a primer binds to itself, reducing effective primer concentration. Primer dimers form when two primers bind each other instead of template. Both waste reagents and reduce yield. The calculator checks for these structures and warns before you run the experiment.
FAQ
What is primer Tm and why does it matter for PCR?
Primer Tm is the temperature at which 50% of primer-template duplexes dissociate. PCR annealing temperature is typically set 3-5°C below Tm. Incorrect Tm estimation leads to no amplification (temperature too high) or non-specific bands (too low).
What is the nearest-neighbor method?
It calculates Tm by summing enthalpy and entropy of each adjacent base pair. More accurate than Wallace rule, especially for longer primers and unusual GC content.
How does salt concentration affect PCR primer Tm?
Higher salt concentrations (Na+, Mg2+) stabilise the DNA duplex and increase Tm. Most PCR buffers use 50mM KCl and 1.5-3.5mM MgCl2. The nearest-neighbor Tm is calculated at a reference salt concentration and then corrected for your actual buffer conditions using the built-in correction in this tool.