1. What is the Q5 Primer Tm Equation?

The Q5 Primer Tm equation calculates the melting temperature of DNA primers for PCR applications. It's specifically optimized for Q5 High-Fidelity DNA Polymerase and provides more accurate Tm predictions than simpler formulas.

2. How Does the Calculator Work?

The calculator uses the Q5 Tm equation:

Where:

• \( Tm \) — Melting temperature (°C)
• \( [Na] \) — Sodium concentration (M)
• \( GC \) — GC content (%)
• \( L \) — Primer length (bases)

Explanation: The equation accounts for salt concentration, GC content, and primer length to predict the temperature at which 50% of the DNA duplex dissociates.

3. Importance of Tm Calculation

Details: Accurate Tm prediction is crucial for PCR optimization, ensuring proper primer annealing temperatures and minimizing non-specific amplification.

4. Using the Calculator

Tips: Enter sodium concentration in molarity (M), GC content as percentage (%), and primer length in bases. All values must be valid (Na > 0, GC between 0-100%, length > 0).

5. Frequently Asked Questions (FAQ)

Q1: Why use Q5 equation instead of Wallace rule?
A: The Q5 equation accounts for salt concentration and provides more accurate Tm predictions, especially for Q5 polymerase.

Q2: What are typical Tm values for PCR primers?
A: Optimal Tm is typically 60-72°C for most applications, with primers within 5°C of each other.

Q3: How does salt concentration affect Tm?
A: Higher salt concentrations stabilize DNA duplexes, increasing Tm. The effect is logarithmic as shown in the equation.

Q4: Why is GC content important?
A: GC pairs have three hydrogen bonds (vs two in AT pairs), making GC-rich sequences more thermally stable.

Q5: Should I use this for non-Q5 polymerases?
A: While developed for Q5, this equation works well for other high-fidelity polymerases but may need adjustment for standard Taq.