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PCR Primer Melting Temperature Formula:
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The melting temperature (Tm) of a PCR primer is the temperature at which half of the DNA duplex dissociates into single strands. It is a critical parameter in PCR optimization, affecting primer annealing efficiency and specificity.
The calculator uses the basic melting temperature formula:
Where:
Explanation: This formula accounts for the different hydrogen bonding strengths between base pairs. GC pairs have three hydrogen bonds and contribute more to thermal stability than AT pairs, which have only two hydrogen bonds.
Details: Accurate Tm calculation is essential for designing effective PCR primers. Proper annealing temperature ensures specific binding to target sequences and minimizes non-specific amplification.
Tips: Enter the count of each nucleotide base in your primer sequence. All values must be non-negative integers. The calculator will compute the melting temperature in degrees Celsius.
Q1: Why are GC bases weighted more heavily than AT bases?
A: GC base pairs form three hydrogen bonds, making them more thermally stable than AT base pairs which form only two hydrogen bonds.
Q2: What is the ideal Tm range for PCR primers?
A: Typically 55-65°C, with forward and reverse primers having Tm values within 5°C of each other for optimal PCR performance.
Q3: Are there more accurate Tm calculation methods?
A: Yes, the nearest-neighbor method and salt-adjusted formulas provide more accurate predictions, especially for longer primers and different salt concentrations.
Q4: How does primer length affect Tm?
A: Longer primers generally have higher Tm values due to increased base pairing and stacking interactions.
Q5: What factors can affect actual Tm in PCR?
A: Salt concentration, primer concentration, DNA template concentration, and the presence of additives like DMSO or formamide can all influence the actual melting temperature.