Oligo Tm calculator
An oligo Tm calculator estimates the melting temperature of a DNA oligonucleotide — the temperature at which half the duplex has dissociated — which is what sets the annealing temperature of a PCR. Talindrew calculates Tm using nearest-neighbour thermodynamics rather than a naive GC rule, and reports GC content and length alongside it. For annealed oligo pairs it also gives duplex Tm and looks up which restriction enzymes leave compatible overhangs.
Specifications
| Method | Nearest-neighbour thermodynamics |
|---|---|
| Reported per primer | Tm, GC content, length |
| Duplex mode | Annealed-pair Tm plus compatible-overhang enzyme lookup |
| Cloning primers | Strategy-specific tails handled separately from the annealing region |
| Detected priming sites | M13, T7, T3, SP6 |
| Price | Free |
Why do Tm calculators disagree?
Two calculators will report different melting temperatures for the same oligo, often by several degrees, and both can be right. The difference comes from the model and its assumptions: a simple Wallace rule counts bases, a GC-percentage formula ignores sequence order entirely, and nearest-neighbour models account for stacking energies between adjacent base pairs. Nearest-neighbour results also depend on the assumed salt and primer concentrations. When you compare a Tm across two tools, check that both use the same model and the same reaction conditions before concluding either is wrong.
Tm for cloning primers with tails
Primers for Gibson, Golden Gate or site-directed mutagenesis carry a 5′ tail that does not bind the template in the first cycles. Calculating Tm across the whole primer, tail included, overestimates the annealing temperature and can cause the reaction to fail. Calculate the Tm on the template-binding portion only, set the annealing temperature from that for the early cycles, and expect the effective Tm to rise once the tail is incorporated into the product.
Practical targets
Reasonable defaults for standard cloning primers.
- Annealing region 18–25 nt.
- Tm around 60 °C, with the primer pair matched to within about 5 °C.
- GC content between 40% and 60%.
- Avoid runs of four or more identical bases and strong 3′ self-complementarity.
Frequently asked questions
- How is melting temperature calculated?
- Talindrew uses nearest-neighbour thermodynamics, which sums the stacking free energies of adjacent base pairs rather than counting GC content. This is substantially more accurate than the Wallace rule or a GC-percentage formula, particularly for primers shorter than 20 nucleotides or with unusual base composition.
- Why does my Tm differ from another calculator?
- Different tools use different models — Wallace, GC-percentage, or nearest-neighbour — and different assumed salt and oligo concentrations. Differences of several degrees between tools are normal and do not mean one is broken. Compare like with like by checking the model and reaction conditions before assuming a discrepancy is an error.
- Should I include the primer tail when calculating Tm?
- No. For cloning primers with a 5′ overhang, calculate Tm on the template-binding region only. The tail does not anneal in the first cycles, so including it overestimates the annealing temperature and is a common cause of failed cloning PCRs.
Related
Try it on your own sequence.
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