1. What is Phusion Annealing Temperature?

The Phusion annealing temperature is typically set 3°C below the melting temperature (Tm) of the primers. Phusion DNA Polymerase allows for higher specificity and efficiency at these slightly lower annealing temperatures compared to other polymerases.

2. How Does the Calculator Work?

The calculator uses the simple formula:

Where:

• \( Ta \) — Annealing temperature in °C
• \( Tm \) — Melting temperature of primers in °C

Explanation: The -3°C adjustment accounts for the optimal working temperature of Phusion DNA Polymerase while maintaining primer specificity.

3. Importance of Correct Annealing Temperature

Details: Proper annealing temperature is crucial for PCR success. Too high may reduce yield, while too low may increase non-specific binding. Phusion's robust performance at Tm-3°C provides both specificity and efficiency.

4. Using the Calculator

Tips: Enter the melting temperature (Tm) of your primers in °C. The Tm can be calculated using various methods (nearest neighbor, basic formula) from your primer sequence.

5. Frequently Asked Questions (FAQ)

Q1: Why subtract 3°C for Phusion polymerase?
A: Phusion DNA Polymerase has different optimal working conditions compared to Taq polymerase, and the -3°C adjustment provides the best balance of specificity and efficiency.

Q2: How do I determine my primer's Tm?
A: Tm can be calculated using primer analysis software or online tools that consider primer length, GC content, and sequence.

Q3: Should I use the same Ta for both primers?
A: If primers have different Tms, use the lower Tm for calculation or consider designing new primers with more similar Tms.

Q4: Can I use this for other polymerases?
A: No, this calculator is specific for Phusion polymerase. Other polymerases may require different temperature adjustments.

Q5: What if my PCR isn't working with this Ta?
A: Consider optimizing in a temperature gradient around the calculated Ta (±2°C) to find the ideal temperature for your specific reaction.