Calculadora de Tm de Primers

Ingresa la secuencia de primer de ADN para calcular Tm usando tres métodos: básico, sal-ajustado y termodinámica de vecino más cercano. Muestra contenido GC, peso molecular y temperatura de annealing sugerida para PCR.

Primer Sequence (5′→3′)

5′-ATGCGATCGATCGTAGC-3′ (17 nt)

Na⁺ Concentration

Primer Concentration

Mg²⁺ Concentration

Melting Temperature (Tm) Results

Basic (Wallace)

52.0°C

2(A+T) + 4(G+C)

Salt-adjusted

46.3°C

Owczarzy

Nearest-Neighbor

51.2°C

SantaLucia 1998

Length:17 nt

GC%:52.9%

MW:4281

Self-complementary:No

AT: 8GC: 9 (52.9%)

All primer quality checks passed

Tm Calculation Methods

Basic (Wallace)— Wallace rule: Tm = 2(A+T) + 4(G+C). Simple estimation for primers ≤14 nt. Does not account for salt or sequence context.

Salt-adjusted— Owczarzy formula: Tm = 81.5 + 16.6×log₁₀([Na⁺]) + 41×(GC/N) − 600/N. Accounts for Na⁺ concentration and primer length.

Nearest-Neighbor— SantaLucia 1998 unified parameters. Uses nearest-neighbor thermodynamics (ΔH, ΔS) for all 16 dinucleotide pairs. Most accurate for typical PCR primers (15–30 nt).

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What is a Primer Tm Calculator?

A primer Tm calculator estimates the melting temperature at which 50% of double-stranded DNA dissociates into single strands. The melting temperature is critical for PCR primer design — optimal annealing temperatures are typically set 5°C below the Tm. This calculator provides three methods: the Basic Wallace rule (2(A+T) + 4(G+C)) for quick estimates, Salt-adjusted (Owczarzy) accounting for Na⁺ concentration, and Nearest-Neighbor (SantaLucia 1998) using thermodynamic parameters (ΔH, ΔS) for all 16 dinucleotide pairs — the most accurate method for 15–30 nt primers.

How to Use This Calculator

Enter your DNA primer sequence (5′→3′ direction, only A/T/G/C bases)
Adjust PCR conditions: Na⁺ concentration (default 50 mM), primer concentration (default 250 nM)
View Tm results from three methods — Nearest-Neighbor (highlighted) is recommended for most primers
Check the GC% bar and primer quality warnings for potential issues
Click the copy button to export all results for your lab notebook

Frequently Asked Questions

Which Tm calculation method should I use for PCR primer design?

For PCR primers of 15–30 nucleotides, the Nearest-Neighbor method (SantaLucia 1998) is the most accurate because it considers the sequence context of every adjacent base pair. The Basic/Wallace rule is only reliable for short oligonucleotides (≤14 nt), and the Salt-adjusted method provides a middle ground when you need a quick estimate that accounts for ionic strength.

What is the ideal GC content for a PCR primer?

The ideal GC content for PCR primers is 40–60%. GC base pairs form three hydrogen bonds (versus two for AT), so higher GC content increases binding stability. Below 40% GC, primer-template binding may be too weak. Above 60%, the primer may form secondary structures (hairpins, dimers) that reduce amplification efficiency.

How does Na⁺ concentration affect primer Tm?

Sodium ions (Na⁺) neutralize the negative charges on DNA phosphate backbone, stabilizing the double helix and increasing Tm. Standard PCR buffers contain about 50 mM Na⁺ equivalent (from KCl and other salts). Higher salt concentrations increase Tm, while lower concentrations decrease it. The Nearest-Neighbor and Salt-adjusted methods both account for this effect.

What does the GC clamp warning mean?

A GC clamp refers to having multiple G or C bases at the 3′ end of the primer. While 1–2 GC bases at the 3′ end promote stable binding to the template, having 4 or more out of the last 5 bases as G/C can cause excessive 3′ stability, leading to nonspecific priming and primer-dimer formation.