PCR Primer Tm Calculator

The melting temperature (Tm) is the temperature at which 50% of DNA primers and their complementary sequences are in a duplex state. It's crucial for determining optimal annealing temperatures in PCR reactions.

Accurate Tm calculation is essential for: • Optimizing PCR conditions • Ensuring specific primer binding • Reducing non-specific amplification • Improving PCR efficiency • Designing multiplex PCR experiments

1. Enter your primer sequence (A, T, C, G only) 2. Input the salt concentration (mM) 3. Click calculate to determine: • Basic Tm (Wallace rule) • Salt-adjusted Tm • GC content • Primer length Note: For best PCR results, aim for: • Primer length: 18-25 nucleotides • GC content: 40-60% • Similar Tm values for primer pairs

• Basic Tm: Simple calculation based on nucleotide composition • Salt-adjusted Tm: Accounts for salt concentration effects • GC Content: Higher GC content generally means higher Tm • Optimal annealing temperature is typically 5°C below Tm

• Avoid long runs of single nucleotides • Check for self-complementarity • Avoid secondary structure formation • Keep GC content balanced • Consider using primer design software for complex applications • Validate primers experimentally