1. What is Phusion Annealing Temperature?

The Phusion annealing temperature is typically set 3°C below the melting temperature (Tm) of the primers. This is a standard recommendation for Phusion DNA Polymerase, known for its high fidelity and processivity.

2. How Does the Calculator Work?

The calculator uses the simple formula:

Where:

• \( T_a \) — Annealing temperature (°C)
• \( T_m \) — Melting temperature of primers (°C)

Explanation: For Phusion DNA Polymerase, setting the annealing temperature 3°C below the Tm of the primers typically provides optimal specificity and yield.

3. Importance of Correct Annealing Temperature

Details: The annealing temperature is critical for PCR success. Too high may result in poor primer binding, while too low may lead to non-specific amplification. Phusion polymerase's robust performance allows this simplified calculation.

4. Using the Calculator

Tips: Enter the melting temperature (Tm) of your primers in °C. The calculator will automatically subtract 3°C to give the recommended annealing temperature for Phusion polymerase.

5. Frequently Asked Questions (FAQ)

Q1: How do I determine the Tm of my primers?
A: Tm can be calculated using the nearest-neighbor method (recommended) or the simple Wallace rule (Tm = 2°C × (A+T) + 4°C × (G+C)).

Q2: Can I use this for other polymerases?
A: No, this calculator is specific to Phusion polymerase. Other polymerases may require different annealing temperature calculations.

Q3: What if my PCR isn't working with this temperature?
A: You may need to optimize further by testing a temperature gradient around the calculated Ta.

Q4: Does primer concentration affect annealing temperature?
A: Yes, higher primer concentrations may allow slightly lower annealing temperatures, but the effect is usually minor.

Q5: What about degenerate primers?
A: For degenerate primers, calculate Tm based on the most stable binding sequence, then subtract 3°C as usual.