Calculate the optimal incubation temperature for thermostable ligase reactions. Nearest-neighbour Tm for upstream and downstream probes with salt correction, mismatch discrimination warning, and support for Taq DNA Ligase, 9°N DNA Ligase, and Ampligase.
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Buffer: 20 mM Tris-HCl pH 7.6, 25 mM KOAc, 10 mM Mg(OAc)2, 1 mM NAD+, 10 mM DTT, 0.1% Triton X-100 | Optimal range: 45–65°C
5′-phosphorylated probe, entered 5′→3′
Downstream of nick site, entered 5′→3′
When to use
Do not use for
For robust mismatch discrimination, design probes so that the perfectly matched Tm is at least 5 °C above the mismatched Tm. The calculator warns you when the probe Tm gap is too narrow for reliable discrimination.
The upstream probe must be 5′-phosphorylated for ligation. Enter it 5′→3′ as it hybridises to the template. The downstream probe hybridises immediately 3′ of the nick site.
Tm drops significantly at low salt. Default buffer [K⁺/Na⁺] 25 mM gives Tm values 10–20 °C lower than the 1 M NaCl reference. Always match the salt input to your actual buffer.
Thermostable ligases use NAD⁺ (not ATP) as a cofactor. This does not affect Tm calculation but is critical for reaction setup — do not substitute ATP.
LDR uses thermal cycling (denature at 95 °C, ligate at recommended T). Padlock probes can use isothermal ligation if probes have sufficiently high Tm. Adjust your protocol accordingly.
Nearest-neighbour Tm calculation using SantaLucia (1998) unified parameters (PNAS 95:1460–1465). Salt correction: ΔS_adj = ΔS + 0.368 × (N−1) × ln[Na⁺]. Recommended temperature = lower probe Tm − enzyme-specific offset (5–7 °C). Mismatch discrimination warning threshold: Tm gap < 5 °C.
Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.
ConductScience Thermostable Ligase Reaction Temperature Calculator (v1.15.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/thermostable-ligase-temp-calculator
SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA. 1998;95:1460–1465. doi:10.1073/pnas.95.4.1460
NEB. Thermostable Ligase Reaction Temperature Calculator. New England Biolabs. 2024. Available at: https://ligasecalc.neb.com/
The nearest-neighbour model predicts DNA duplex stability by summing thermodynamic contributions of each overlapping dinucleotide pair (e.g. 5′-AG-3′ stacked on 3′-TC-5′). The SantaLucia (1998) unified parameters provide ΔH and ΔS for all 10 unique Watson–Crick dinucleotides.
Where ΔH is in cal/mol, R = 1.987 cal/(mol·K), and Ct is the total strand concentration in molar. This gives the Tm at 1 M NaCl; the salt correction adjusts ΔS for the actual buffer ionic strength.
Monovalent cations stabilise DNA duplexes by neutralising the phosphate backbone charge. At lower salt concentrations, electrostatic repulsion destabilises the duplex and lowers Tm. The SantaLucia (1998) correction:
This adds a per-base-pair entropy term proportional to ln[Na⁺]. At 1 M NaCl (ln = 0), the correction vanishes. Typical ligase buffers contain 25 mM KOAc or KCl, giving a significant reduction in Tm compared to the 1 M standard.
In a ligase detection reaction (LDR) or oligonucleotide ligation assay (OLA), two probes hybridise adjacently on a target template. The ligase seals the nick only when there is perfect complementarity at the junction. Temperature must be:
1. High enough to melt mismatched probe–template duplexes (discrimination) 2. Low enough that perfectly matched probes stay hybridised (sensitivity)
The sweet spot is typically 5–10 °C below the lower probe Tm. Probes should be designed with similar Tm values and a Tm gap 5 °C above the mismatch Tm for reliable single-base discrimination.
