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NEB Tm Calculator — PCR Primer Melting Temperature | Calcgator

NEB Tm Calculator

Calculate PCR primer melting temperature for Q5, OneTaq, Taq, Phusion, LongAmp and more. Real-time Tm and annealing temperature using NEB-specific buffer corrections, SantaLucia nearest-neighbour thermodynamics, and built-in primer quality checks. No signup. Free forever.

⚡ Real-time results 🔬 6+ polymerases ✓ Primer quality checks 📱 Mobile-friendly
Tm (Q5) 62°C Annealing 59°C
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NEB Tm Calculator
Primer melting temperature with polymerase-specific corrections
Polymerase
Forward Primer (5′→3′)
Reverse Primer (5′→3′)
+
Advanced settings (salt & primer concentration)
NEB buffers typically 50–100 mM Na⁺ equivalent
Typical range: 200–500 nM for standard PCR
Recommended Annealing Temperature
—°C
🔬 Q5® High-Fidelity
● Forward Primer
—°C
● Reverse Primer
—°C
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Primer Tm range on temperature scale
Low
Ideal
High
40°C50°C60°C70°C80°C
—
Lower Tm
—
ΔTm
—
Poly offset
Formula: SantaLucia 1998 NN + NEB correction
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Enter primer sequences above for annealing temperature recommendations.
📋 Polymerase Cheat Sheet
Polymerase Ta offset Fidelity
Q5®Tm − 3°CHigh
OneTaq®Tm − 4°CStandard
TaqTm − 5°CStandard
Phusion®Tm − 3°CHigh
LongAmp®Tm − 4°CStandard
Vent®Tm − 4°CHigh
Ta = annealing temperature. Always run a gradient PCR ±5°C around recommended Ta to empirically optimise.
✅ Primer Design Rules
📏
Length: 18–25 bp
Shorter = lower Tm, longer = higher secondary structure risk
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GC content: 40–60%
Ideal Tm range 55–65°C for most polymerases
⚓
GC clamp: end in G or C
Anchors 3′ end to template for efficient extension
🌡️
ΔTm ≤ 5°C between pair
Large mismatch reduces amplification efficiency
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Avoid runs of ≥4 same base
e.g. AAAA or GGGG causes slippage and mispriming
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The science behind Tm calculation

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SantaLucia 1998 NN method

How Primer Tm Is Calculated

This calculator uses the SantaLucia 1998 nearest-neighbour thermodynamics model — the gold standard for primer Tm prediction. Each adjacent base-pair combination (AA/TT, AT/TA, GC/CG etc.) contributes known ΔH and ΔS values. The Tm is then: Tm = ΔH / (ΔS + R·ln(Cp/4)) − 273.15, where Cp is primer concentration and R is the gas constant.

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Buffer-specific salt corrections

Why NEB Tm Differs From Other Calculators

NEB's buffers have unique salt compositions that affect DNA duplex stability. Q5® buffer results in a higher effective Tm (+3.5°C vs standard). This calculator applies NEB-specific corrections using Owen & Owczarzy's salt correction formula: 16.6 × log₁₀([Na⁺]), then subtracts a polymerase-specific annealing offset (Q5: −3°C, Taq: −5°C, Phusion: −3°C).

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GC clamp + mismatch detection

Primer Quality Checks Explained

Beyond Tm, primer success depends on quality factors most calculators ignore. This tool checks: GC clamp (3′ end must be G or C for firm template anchoring), base runs (≥4 of same base causes slippage), Tm mismatch (>5°C between primer pairs reduces efficiency), and palindrome risk (sequences that can self-fold into hairpin structures that block extension).

FAQ

Everything about
NEB Tm calculation

Common questions about primer Tm, Q5 polymerase, and PCR annealing temperature — answered below.

💬 Ask us anything
This calculator uses SantaLucia 1998 nearest-neighbour thermodynamics — the gold standard for primer Tm prediction. Each adjacent base-pair combination contributes specific ΔH (enthalpy) and ΔS (entropy) values from the published lookup table. The raw Tm is then corrected for your actual salt concentration and primer concentration using the Owczarzy salt correction formula. Finally, a polymerase-specific annealing offset is applied (Q5: −3°C, Taq: −5°C) to give you a practical annealing temperature recommendation.
For Q5® polymerase, NEB applies an additional +3.5°C buffer adjustment on top of the SantaLucia nearest-neighbour Tm. This accounts for Q5's unique high-fidelity buffer composition which stabilises DNA duplexes more than standard buffers. The recommended annealing temperature is then set 3°C below the corrected Tm. Example: if your calculated Tm is 65°C with Q5 buffer correction, your recommended annealing temperature is 62°C.
Most basic Tm calculators use the simple Wallace rule (Tm = 2(A+T) + 4(G+C)) or a fixed salt concentration, which ignores your actual buffer conditions. This tool uses nearest-neighbour thermodynamics with real salt correction, which gives 1–3°C more accurate results. Additionally, NEB polymerase-specific buffer corrections account for the fact that Q5 buffer has higher effective salt content than standard Taq buffers, raising the effective Tm.
A GC clamp means the last 1–2 bases at the 3′ end of the primer are G or C. G-C base pairs form 3 hydrogen bonds (vs 2 for A-T), so having G or C at the 3′ end firmly anchors the primer to the template during the extension step. Without a GC clamp, the primer's 3′ end can "breathe" or melt off before polymerase can extend it, dramatically reducing PCR efficiency. This calculator flags primers missing a GC clamp.
Ideal PCR primers have 40–60% GC content. Below 40%: the Tm drops, primers bind weakly, and you get low yield or no product. Above 70%: primers are prone to forming secondary structures (hairpins) and can cause non-specific binding at multiple template locations. For GC-rich templates (>65% GC), you may need to add DMSO or betaine to your reaction to destabilise secondary structures.
Enter your forward primer in the first box and reverse primer in the second box (both 5′→3′). Select your polymerase. Results update in real-time — you'll see Tm for each primer individually, plus the recommended annealing temperature for the pair (based on the lower Tm minus the polymerase offset). If the two Tms differ by more than 5°C, the calculator warns you that amplification efficiency may be reduced.