1. What is PCR Annealing Temperature?

The annealing temperature (Ta) is the temperature at which primers bind to the template DNA during PCR. Optimal Ta is critical for specific and efficient amplification, typically 3-5°C below the primer melting temperature (Tm).

2. How Does the Calculator Work?

The calculator uses the following approach:

Where:

• \( T_m \) — Melting temperature (°C)
• \( G+C \) — Number of G and C nucleotides
• \( A+T \) — Number of A and T nucleotides
• \( \Delta T \) — Polymerase-specific adjustment (1-5°C)

3. Importance of Annealing Temperature

Details: Correct Ta ensures primer specificity and amplification efficiency. Too high may reduce yield; too low may cause non-specific binding.

4. Using the Calculator

Tips: Enter primer sequence (5'→3'), select polymerase type and salt concentration. The calculator provides Tm and recommended Ta.

5. Frequently Asked Questions (FAQ)

Q1: Why different Ta for different polymerases?
A: Polymerases have different processivities and optimal temperature ranges (e.g., Q5 works best at higher Ta than Taq).

Q2: What if my PCR doesn't work with calculated Ta?
A: Try a temperature gradient (±2-3°C from calculated Ta) to optimize.

Q3: How does salt concentration affect Ta?
A: Higher salt increases Tm by stabilizing DNA duplexes (add ~5°C for high salt buffers).

Q4: Should I use the same Ta for both primers?
A: For primers with different Tm, use the lower Tm to calculate Ta or design new primers with matched Tm.

Q5: What about degenerate primers?
A: This calculator is for defined sequences. For degenerate primers, calculate Tm for each possible sequence and use the lowest.