Oligo Primer Melting Temperature (Tm), GC% & Annealing Temp Calculator

Compute primer length, GC%, basic & salt-adjusted Tm (°C), and annealing temperature.

Basic Primer Melting Temperature (T_m)
53.83 °C
Recommended Annealing Temp (T_m − 5°C) 48.83 °C
GC Content Percentage 55 %
Oligo Length (Nucleotides) 20 nt
Inputs
bases
Number of Guanine (G) bases in primer.
bases
Number of Cytosine (C) bases in primer.
bases
Number of Adenine (A) bases in primer.
bases
Number of Thymine (T) bases in primer.
Results & Graph
Basic Primer Melting Temperature (T_m)
53.83 °C
Recommended Annealing Temp (T_m − 5°C)
48.83 °C
GC Content Percentage
55 %
Oligo Length (Nucleotides)
20 nt

Basic Primer Melting Temperature (T_m)

Graph 1 • Count G Sensitivity Recalculates the domain equation across 50% to 150% of the primary input while holding other parameters constant.
0 °C 32.7 °C 65.5 °C 50%: 44.64 °C 44.6 50% 75%: 49.61 °C 49.6 75% 100%: 53.83 °C 53.8 100% 125%: 57.46 °C 57.5 125% 150%: 60.62 °C 60.6 150%
Graph 2 • Primary & Supporting Metric Comparison Compares the primary output alongside supporting domain benchmarks.
Basic Primer Melting Temperature (T_m) 53.83 °C Recommended Annealing Temp (T_m − 5°C) 48.83 °C GC Content Percentage 55 % Oligo Length (Nucleotides) 20 nt
Graph 3 • Exact Output Table & Units Displays each calculated output in its exact domain unit.
Basic Primer Melting Temperature (T_m)53.83 °CRecommended Annealing Temp (T_m − 5°C)48.83 °CGC Content Percentage55 %Oligo Length (Nucleotides)20 nt
Summary: A 20-nt oligo (55% GC) has T_m = 53.8°C (Wallace T_m = 62°C) and recommended annealing temperature T_a ≈ 48.8°C.
Summary: A 20-nt oligo (55% GC) has T_m = 53.8°C (Wallace T_m = 62°C) and recommended annealing temperature T_a ≈ 48.8°C.
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Formula & Mathematical Methodology: Primer Tm & GC% Reference Equation

GC% = [(G + C) / N] × 100
Tm = 64.9 + 41 × (G + C − 16.4) / N
GC% = [(G + C) / N] × 100; T_m = 64.9 + 41 × (G + C − 16.4) / N
  • countG: Count G (bases)
  • countC: Count C (bases)
  • countA: Count A (bases)
  • countT: Count T (bases)

Rounding: Laboratory and clinical equations retain full floating-point precision and round displayed results to 2 decimal places.

Worked Example: Reference Calculation: Primer Tm & GC%

Compute primer length, GC%, basic & salt-adjusted Tm (°C), and annealing temperature.

  1. Enter the reagent concentrations, assay readings, or clinical laboratory parameters.
  2. Compute the standard formula: GC% = [(G + C) / N] × 100; T_m = 64.9 + 41 × (G + C − 16.4) / N.
  3. Review the primary result, supporting reference intervals, and 3 SVG visualizations.
Result: Evaluates Primer Tm & GC% via GC% = [(G + C) / N] × 100; T_m = 64.9 + 41 × (G + C − 16.4) / N.

Frequently Asked Questions

What formula is used by the Oligo Primer Melting Temperature (Tm), GC% & Annealing Temp Calculator?

It computes GC% = [(G + C) / N] × 100; T_m = 64.9 + 41 × (G + C − 16.4) / N and plots three custom SVG graphs showing parameter sensitivity and component breakdowns.

Can I compare different reagent dilutions or lab scenarios side by side?

Yes. Use the "Compare (0/3)" button to pin up to 3 scenarios side by side.

Are entered values stored on any server?

No. Every calculation runs 100% locally in your browser.

Science & Engineering · Molecular Biology & Genetics

Oligo Primer Melting Temperature (Tm), GC% & Annealing Temp Calculator: inputs, method, and how to read the result

Compute primer length, GC%, basic & salt-adjusted Tm (°C), and annealing temperature.

Choose inputs for the case you are checking

The defaults are illustrative. Replace them with values for your case and keep the units shown beside each field.

  • Count G: Number of Guanine (G) bases in primer.
  • Count C: Number of Cytosine (C) bases in primer.
  • Count A: Number of Adenine (A) bases in primer.
  • Count T: Number of Thymine (T) bases in primer.

What the calculation includes

With the default values shown, Basic Primer Melting Temperature (T_m) is 53.83 °C. The calculation follows Primer Tm & GC% Reference Equation; the formula and variable definitions above show how the inputs combine.

One-input check: With the other defaults held constant, changing Count G from 6 bases to 7 bases changes Basic Primer Melting Temperature (T_m) from 53.83 °C to 56.31 °C. This is a comparison inside the calculator's stated model, not a prediction.

Where to use caution

  • Uses standard IUPAC atomic weights, 25°C (298.15 K) reference thermodynamics, and published clinical literature equations.
  • Educational and laboratory reference tool only; clinical scoring calculators do not replace professional medical judgment or institutional protocols.

Scope: IUPAC / NIH Laboratory & Clinical Reference Standard Check the assumptions and limitations against your situation before relying on the estimate.

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