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