PCR Primer Tm Calculator
Calculates the melting temperature (Tm) of short PCR primers using the Wallace rule base-counting method. Use it when designing primers under 20 bases or for a quick sanity check before ordering oligos.
About this calculator
The melting temperature (Tm) is the temperature at which 50% of a DNA duplex is single-stranded. For short oligonucleotides (typically ≤20 bases), the Wallace rule provides a fast estimate: Tm = 2 × (A + T) + 4 × (G + C), where A, T, G, and C are the counts of each respective base in the primer. The logic behind the formula is that each A–T base pair contributes 2°C of stability (held by 2 hydrogen bonds), while each G–C pair contributes 4°C (held by 3 hydrogen bonds). Longer primers and high-GC sequences require more sophisticated nearest-neighbor thermodynamic models, but the Wallace rule remains a reliable starting point for routine primer design.
How to use
Suppose your primer sequence is 5'-AATGCGGCTAC-3' (11 bases): A = 3, T = 2, G = 3, C = 3. Step 1 — Enter 3 in 'Adenine Count'. Step 2 — Enter 2 in 'Thymine Count'. Step 3 — Enter 3 in 'Guanine Count'. Step 4 — Enter 3 in 'Cytosine Count'. Step 5 — The calculator computes: Tm = 2 × (3 + 2) + 4 × (3 + 3) = 2 × 5 + 4 × 6 = 10 + 24 = 34°C. This means your PCR annealing temperature should be set a few degrees below 34°C for this primer.
Frequently asked questions
When should I use the Wallace rule Tm formula versus a nearest-neighbor model?
The Wallace rule (Tm = 2(A+T) + 4(G+C)) is accurate enough for primers shorter than about 20 bases and is the standard quick-check method in most labs. For primers longer than 20 bases, or when working in non-standard salt concentrations or with modifications like locked nucleic acids, nearest-neighbor thermodynamic models are more reliable. Software tools such as Primer3 or OligoCalc implement these advanced models. Use this calculator for rapid comparisons or when designing degenerate primers.
Why do G-C base pairs contribute more to primer melting temperature than A-T pairs?
Guanine and cytosine form three hydrogen bonds with each other, whereas adenine and thymine form only two. This extra hydrogen bond makes G–C pairs significantly more thermally stable, requiring more energy (higher temperature) to denature. A primer rich in G–C content will therefore have a higher Tm and will remain annealed at higher cycling temperatures. The Wallace rule captures this by assigning 4°C per G–C pair versus 2°C per A–T pair.
How do I choose the right PCR annealing temperature based on Tm?
A common rule of thumb is to set the annealing temperature 5°C below the calculated Tm of the lower-melting primer in a pair. For example, if one primer has Tm = 58°C and the other 62°C, use an annealing temperature of about 53°C to start. Too low an annealing temperature increases non-specific binding and spurious bands; too high reduces yield or prevents annealing entirely. Gradient PCR across a range of ±5°C around your estimated annealing temperature is the most reliable way to optimise amplification.