PCR Annealing Temperature Calculator
Explore primer binding. Plan your PCR gradient.
Primer & Reaction Inputs
Annealing Summary
Standard Taq · test empirically
Primer pair review
Tm proximity alone does not establish specificity.
Sequence estimates
| Primer | Length & GC | Estimated Tm |
|---|---|---|
| Forward | — | — |
| Reverse | — | — |
Direct Tm values · no sequence or salt analysis applied
Q5 / Phusion: verify polymerase-specific guidance.
PCR Thermal Cycle Visualizer
Move over or tap the profile to inspect temperature.
One cycle including modelled ramps
Illustrative one-cycle profile · timing and ramp rate are configurable. No amplification yield or specificity is predicted.
Primer Binding Preview
Forward primerReverse primerConceptual binding illustration · not a specificity or alignment result
Calculation & Worked Example
Sequence model: perfect-match DNA nearest-neighbor parameters (DNA_NN3 convention), terminal contributions and self-complementarity correction; entropy-based salt adjustment. Tm = 1000ΔH / (ΔSadjusted + R ln Ceff) − 273.15, R=1.987 cal/(mol·K), Ceff approximated by primer concentration in mol/L with negligible target.
Frequently Asked Questions
How is the starting Ta estimated?
The tool subtracts the entered offset from the lower primer Tm. Positive offsets lower Ta; negative offsets raise it. The chosen half-width defines a gradient centered on that estimate. This is a starting experiment-planning estimate, not an experimentally established optimum.
What changes in sequence mode?
Each primer's length and GC percentage are measured from its A/C/G/T sequence, and a perfect-match nearest-neighbor Tm is estimated under the entered conditions. The lower estimated Tm then enters the same offset calculation. Direct Tm mode uses entered temperatures without applying sequence or salt settings.
Why do Q5 and Phusion differ?
Polymerase and buffer formulations affect suitable annealing conditions. NEB recommends its product-specific calculator. Phusion guidance also distinguishes primer lengths in its protocol. The High Fidelity card here loads a generic zero-offset starting scenario and 98°C denaturation; it does not duplicate either kit's validated calculator.
Does this test primer specificity?
No. Tm, GC and length do not establish whether a primer pair uniquely amplifies a target. This tool does not query genomes, align primers to a template, or evaluate mismatches, hairpins, cross-dimers or off-target amplicons. Use Primer-BLAST and appropriate design tools, then validate experimentally.
More questions (8)
How should I enter primer sequences?
Enter both primers in their own 5′ to 3′ orientation. Spaces and simple terminal 5′/3′ notation are accepted. Use only the template-binding segment: remove adapters, restriction sites and noncomplementary 5′ tails. Ambiguous bases, RNA U and sequences shorter than 8 or longer than 80 nt are rejected rather than silently guessed.
Which thermodynamic model is used?
The model uses the DNA_NN3 perfect-match DNA/DNA parameter convention, including terminal A/T and G/C terms and the entropy penalty for fully self-complementary sequences. It adds 0.368(N−1) ln([salt] in mol/L) to entropy. This is not a mismatch, dangling-end or whole-amplicon melting calculation.
What does primer concentration mean here?
The entered concentration is the molar concentration of each primer in the reaction. The calculation uses a primer-excess approximation and neglects complementary target concentration, so effective concentration in the logarithmic term is approximated by the entered primer concentration. Equimolar duplex experiments require a different concentration treatment.
How are magnesium and dNTPs handled?
If magnesium exceeds total dNTP concentration, its contribution is approximated through a sodium-equivalent term: Na+K + 120√max(Mg−dNTP,0), with inputs in mM. This is an approximate salt treatment, not an equilibrium free-magnesium calculation or a proprietary polymerase-buffer model. DMSO, betaine, Tris, mismatches and other additives are excluded.
What does the thermal graph show?
It shows one user-configured denaturation hold, a linear ramp to Ta, an annealing hold, a linear ramp to extension, and an extension hold. Ramp duration is the absolute temperature difference divided by ramp rate. The time axis includes these ramps. It excludes initial heating, cycle repeats, final extension and storage hold.
Are the defaults a complete PCR protocol?
No. The default 95/57/72°C profile is illustrative. Many actual Taq formulations use 68°C extension, while other products use different temperatures and timing. Use your polymerase instructions, template and amplicon length to set the actual program. A zero-offset High Fidelity preset is not an NEB recommendation.
Does the GC-rich preset account for my template?
No. It labels an optimization context and loads a starting profile; it does not know template GC content or secondary structure. Its illustrative scenario widens the gradient half-width to 4°C and sets a 60 s denaturation hold; these are adjustable examples, not an optimized protocol. Primer GC can be calculated in sequence mode, but template GC is different. No automatic DMSO correction or GC-enhancer dose is inferred.
Why can another Tm calculator disagree?
Parameter tables, concentration conventions, salt models, magnesium binding, additives and kit-specific empirical corrections can all change Tm or suggested Ta. Compare the same model and reaction conditions. Confirm an appropriate gradient experimentally; this tool is not evidence that a PCR will succeed.
Sources & References
NCBI Primer-BLASTPrimer design and specificity screening
NEB PCR OptimizationPolymerase-specific cycling guidance
Method details: SantaLucia (1998) · Thermodynamic conventions · No institutional endorsement implied.
Estimate a starting annealing temperature from primer Tm values or perfect-match DNA nearest-neighbor estimates, and explore a configurable one-cycle thermal profile.