1. What is PCR Melting Temperature?

The melting temperature (Tm) is the temperature at which 50% of the DNA duplex dissociates into single strands. It's a critical parameter in PCR (Polymerase Chain Reaction) for determining optimal annealing temperatures.

2. How Does the Calculator Work?

The calculator uses the basic melting temperature equation:

Where:

• \( G \) — Guanine count in the DNA sequence
• \( C \) — Cytosine count in the DNA sequence
• \( A \) — Adenine count in the DNA sequence
• \( T \) — Thymine count in the DNA sequence

Explanation: The equation accounts for the fact that G-C base pairs (with three hydrogen bonds) are more thermally stable than A-T pairs (with two hydrogen bonds).

3. Importance of Tm Calculation

Details: Accurate Tm calculation is crucial for designing PCR primers, determining optimal annealing temperatures, and ensuring specific and efficient DNA amplification.

4. Using the Calculator

Tips: Enter the count of each nucleotide in your DNA sequence. All values must be non-negative integers. For primers, use the sequence of the primer itself.

5. Frequently Asked Questions (FAQ)

Q1: Is this the most accurate Tm calculation method?
A: This is the basic calculation. More sophisticated methods account for salt concentration, DNA concentration, and nearest-neighbor thermodynamics.

Q2: What's a typical Tm range for PCR primers?
A: Most primers work well with Tm between 50-65°C, with optimal annealing temperature typically 3-5°C below the Tm.

Q3: Why do G and C contribute more to Tm than A and T?
A: G-C pairs have three hydrogen bonds compared to two in A-T pairs, making them more thermally stable.

Q4: Should I use this for long DNA sequences?
A: This simple formula works best for oligonucleotides (18-30 bases). For longer sequences, more complex calculations are needed.

Q5: How does salt concentration affect Tm?
A: Higher salt concentrations increase Tm by stabilizing the DNA duplex. Specialized calculators can account for this.