Tm / Annealing Temperature Calculator
Calculate primer Tm and suggested annealing temperature for single pairs or batch CSV, with QC flags and exportable results.
What it does
Paste forward and reverse primer sequences—or upload a CSV of up to 200 pairs—and pick your polymerase (Q5, Phusion, Taq, KAPA HiFi, or custom salt conditions). Add DMSO or betaine if your PCR mix includes them.
The tool returns Tm for each primer, ΔTm between partners, a suggested annealing temperature, GC%, and Go/Flag/Fail QC for hairpins and dimers. Batch runs produce a sortable table you can export to CSV, copy, or print. A method comparison panel explains why other calculators gave different numbers. Invalid bases are flagged as you type. Everything runs in your browser—sequences never leave your machine.
Why researchers use it
- Process up to 200 primer pairs in one batch run
- Keep proprietary sequences on your machine only
- Adjust Ta for DMSO and betaine in your mix
- Catch unbalanced Tm and dimer issues before PCR
- Compare why vendor calculators disagree on Tm
Best for
- Multiplex and panel PCR primer design
- NGS amplicon primer QC
- GC-rich reactions with DMSO or betaine
- Gradient PCR planning for new primer sets
- Core facility primer order review
What makes it different
NEB Tm Calculator is trusted but limited to NEB enzymes and sends sequences to their servers. IDT OligoAnalyzer checks hairpins and dimers but handles one primer at a time and has no DMSO correction. Labs often use both tools plus Excel loops for batch work.
This browser tool combines batch Tm and Ta for Q5, Phusion, Taq, KAPA, or custom buffers with hairpin and dimer QC, DMSO and betaine adjustments, and pass/fail flags per pair—without uploading sequences. A side-by-side method comparison explains cross-tool Tm differences. Results export to CSV or print for lab notebooks.