Calculadora de Temperatura de Anelamento (PCR)
- Created by
- Renato Passos, Eng. de Software
- Reviewed by
- Renato Passos, Eng. de Software
Last updated: Apr 18, 2026
Formula
Tm = 4(G+C) + 2(A+T); Ta = Tm − 5 °C
About this calculator
The PCR Annealing Temperature Calculator helps estimate the ideal annealing temperature (Ta) for primers based on the simplified Wallace formula. The melting temperature (Tm) is calculated as Tm = 4*(G+C) + 2*(A+T), where G, C, A, and T are the counts of each base in the primer. Then, the suggested Ta is Tm - 5 °C, a common starting point for PCR reactions.
This tool is useful for students and professionals who need a quick starting point for PCR optimization. The correct annealing temperature is crucial for reaction specificity; too low Ta can cause non-specific annealing, while too high Ta reduces efficiency. The calculator handles primers up to 30 bases; for longer primers or extreme compositions, more precise methods are recommended.
Use the calculator when designing primers for conventional PCR, real-time PCR, or other applications requiring precise annealing. Note that the Wallace formula is an approximation and does not account for salt concentration or DMSO. The calculated Ta should be validated experimentally, adjusting as needed.
Frequently asked questions
Why is the annealing temperature Tm - 5 °C?
Tm - 5 °C is a common starting point that balances specificity and efficiency in PCR. The actual temperature should be optimized experimentally.
Does the formula work for long primers?
The Wallace formula is more accurate for primers up to 30 bases. For longer primers, use methods like Nearest Neighbor.
What if the calculated Ta does not work?
Perform a temperature gradient (vary ±5 °C around Ta) to find the best condition. Also adjust MgCl2 concentration.
How does base composition affect Tm?
GC pairs have three hydrogen bonds, increasing Tm. Higher GC content leads to higher Tm and suggested Ta.
Does the calculator consider salt or DMSO?
No. The simple formula ignores reaction components. For more precise calculations, use specialized software that accounts for these factors.