NEB/IDT Primer Tm Calculator with Salt, Mg and Batch Settings

Start with sequence inputCompare methods

Enter a DNA or RNA sequence, match the salt, Mg2+, dNTP, DMSO, formamide, and oligo concentration settings you plan to use, then calculate Tm, ΔH, ΔS, ΔG, suggested annealing temperature, and batch output for up to 1000 sequences.

Browser calculation by defaultSantaLucia + Owczarzy methodsBatch mode up to 1000 sequences

Matching NEB-style, IDT-style, Twist-style, or Q5 conditions? Calculate here after mirroring the reaction assumptions. Need more than Tm? Open the Primer Analyzer for GC, MW, hairpin, and dimer checks. Need to understand a disagreement? Read why Tm calculators disagree.

Input Parameters

Length: 0 nt

Determines which nearest-neighbor thermodynamic parameters to use.

Salt Conditions

Enter sodium ion concentration in millimolar (mM), typically 50-200 mM for PCR

Enter magnesium ion concentration in millimolar (mM), typically 1.5-3 mM for PCR, or 0 if not applicable

Enter dNTP concentration in millimolar (mM), typically 0.2-0.5 mM for PCR, or 0 if not applicable

Other Conditions

Enter oligonucleotide concentration in nanomolar (nM), typically 250-500 nM for PCR

Enter DMSO percentage, typically 0-10% to reduce secondary structure, or 0 if not applicable

Results

No results yet

Enter a sequence and click "Calculate Tm"

What the calculator evaluates: Direct calculator for sequence-specific primer Tm results

Calculations run in the browser by default, and Dashboard History may store an input snapshot when auto-save, session, or account history is enabled. Method references include SantaLucia 1998 nearest-neighbor parameters plus Owczarzy-style salt correction papers. Use this page for the actual primer melting temperature result, then use the NEB, IDT, method-comparison, and research pages for method notes.

If your search started with NEB, IDT, Twist, Q5, or a vendor-style primer Tm workflow, enter the sequence here and mirror the reaction assumptions before comparing results. Keep the vendor calculator in the loop when the final annealing temperature depends on a specific polymerase kit or buffer recommendation.

Free primer Tm calculator using SantaLucia/Owczarzy math with NEB-style settings guidance for a browser-based NEB-style settings workflow. Public calculations run in the browser by default, while optional History saves can store input snapshots when enabled. Keep NEB in the loop when enzyme-specific annealing guidance depends on Q5, Phusion, Taq, OneTaq, or a specific NEB buffer.

Tm Calculation Method Comparison

Enter your sequence and see how 5 different Tm calculation methods produce different results. Understand which method matches NEB, IDT, or Primer3 — and why the differences matter for your experiment.

DNA only (A, T, C, G). Minimum 6 nt.

These conditions affect NN methods. Wallace ignores salt. %GC uses Na⁺ only.

Use This Page for the Actual Tm Calculation

Use this page when you need to calculate primer melting temperature, match NEB/IDT/Twist-style reaction assumptions, account for salt, Mg²⁺, dNTP, DMSO, formamide, or concentration settings, or produce browser-default batch Tm output. Guides explain why values differ; comparisons help you choose a tool or method before calculating.

Result reading on this page stays brief: enter the sequence, mirror the buffer assumptions you actually plan to use, read the Tm and suggested annealing-temperature starting point, then move to the guide or comparison page only if the result needs interpretation.

The calculator uses SantaLucia 1998 nearest-neighbor thermodynamics with Owczarzy-style monovalent correction and a Mg correction after dNTP subtraction; it is not a full vendor-equivalent mixed-cation model. The calculation screen runs locally in your browser, while Dashboard History may store an input snapshot when auto-save, session, or account history is enabled. Open the melting-temperature guide when the question is why tools disagree.

How to Use the Tm Calculator

  1. Enter your oligonucleotide sequence (5' to 3') in the input field. Single-sequence mode accepts DNA bases A, T, C, G or RNA bases A, U, C, G; DNA:RNA hybrid mode uses hybrid parameters, while RNA:RNA-specific thermodynamics are not implemented.
  2. Set the reaction conditions: Na⁺ concentration (default 50 mM), Mg²⁺ concentration (default 0 mM), and oligonucleotide concentration (default 250 nM). Match these to your actual PCR buffer.
  3. If using DMSO or formamide, enter the percentage in the correction fields.
  4. For multiple primers, switch to Batch Mode and paste one sequence per line or upload a FASTA file.
  5. Click "Calculate" to see results including Tm, salt-corrected Tm, suggested annealing temperature, GC content, sequence length, and thermodynamic parameters (ΔH, ΔS, ΔG).
  6. Use the suggested annealing temperature (Ta = Tm - 5°C) as a starting point for your PCR optimization.

Frequently Asked Questions

Which settings should I enter for a method-based Tm calculation?
Enter the primer sequence first, then match the reaction conditions you will use in the lab: Na⁺, Mg²⁺, dNTP concentration, DMSO or formamide percentage, and oligo concentration. If you are checking NEB-style, IDT-style, Twist-style, or Q5 conditions, use this page for the actual Tm calculation and mirror the buffer assumptions as closely as possible.
How do I mirror NEB, IDT, or Twist-style Tm settings?
Start with the exact primer sequence, then copy the assumptions that drive the vendor result as closely as this calculator exposes them: polymerase or buffer family, Na⁺, Mg²⁺, dNTP, DMSO or formamide, and primer concentration. Use NEB or the vendor page when enzyme-specific annealing guidance is required; use this calculator when you need browser-default batch Tm, explicit salt settings, and related primer context in one workflow.
What method does this free Tm calculator use?
The result depends on the algorithm, thermodynamic parameters, and buffer assumptions used. This calculator uses published SantaLucia nearest-neighbor parameters with Owczarzy-style monovalent and Mg corrections after dNTP subtraction, then keeps the calculation itself client-side; Dashboard History may store an input snapshot when saving is enabled.
When should I use the comparison pages instead of this calculator?
Use this page when you already need a Tm number. Use the NEB, IDT, or method-comparison pages when the question is which method or buffer assumption to use before calculating. After that decision is made, return here with the sequence and actual salt, Mg²⁺, dNTP, DMSO, and concentration settings.
Can I calculate Tm for many primers at once?
Yes. Switch to Batch Mode, paste one sequence per line or upload FASTA-style input, then use the same buffer assumptions across the set. Batch mode is the right path when you need to screen primer panels, compare Tm windows, or prepare a list for downstream QC.

Related Tools

Primer Analyzer

OligoAnalyzer-style primer review with Tm, GC%, self-complementarity, and dimer checks.

Tm Methods Comparison

Side-by-side comparison of SantaLucia, Owczarzy, Wallace, and %GC methods.

GC Content Analyzer

Analyze GC percentage and base composition. Identify extreme GC content that may affect Tm accuracy.

Secondary Structure Predictor

Predict hairpins and dimers that compete with primer-template binding and affect effective Tm.

NEB Tm Calculator Comparison

Use this support page when you need NEB-specific polymerase and buffer context before calculating.

Tm Calculator Alternatives

Choose between OligoPool, NEB, IDT, Twist, Thermo, Sigma, and other Tm workflows before calculating.

Annealing Temperature from Primer Tm

Turn calculated primer Tm values into a PCR annealing-temperature starting point.

Free Oligo Tool Comparison

Compare online tools for Tm calculation, primer analysis, structure checks, and batch QC.

Review request

Check a result or ordering detail

Send the calculation, settings, or pool submission detail that needs a second look.

Related reading

Continue with the page or tool that matches the next decision in your experiment.

Accuracy Validation: OligoPool vs NEB & IDTNEB Tm Calculator ComparisonIDT OligoAnalyzer ComparisonTm Calculator AlternativesAnnealing Temperature from Primer TmSalt Correction Method ComparisonΔG Threshold Database — Predict PCR SuccessTm Method ReviewValidate PCR Primers Before OrderingTm Method ReferenceQuick Answers for Tm Decisions