Primer Tm Calculator
Calculate melting temperature for PCR primers using multiple methods. Essential for primer design and PCR optimization.
✓ Multiple calculation methods✓ Salt concentration adjustment✓ Annealing temperature suggestion
Primer Sequence & Conditions
0 bases
Understanding Primer Melting Temperature
What is Tm?
Melting temperature (Tm) is the temperature at which 50% of primer molecules are bound to their target sequence.
- • Higher Tm = more stable binding
- • Affected by sequence composition
- • Influenced by salt concentration
- • Critical for PCR success
Calculation Methods
Wallace Rule:
Quick estimate: 2°C per A/T, 4°C per G/C
GC Content:
Based on %GC and primer length
Nearest Neighbor:
Most accurate, considers base stacking
Primer Design Guidelines
Ideal Tm Range
• 50-65°C for most applications
• Forward and reverse within 5°C
• Higher for longer amplicons
Primer Length
• 18-24 nucleotides typical
• Shorter = less specific
• Longer = higher Tm
GC Content
• 40-60% optimal range
• Avoid long GC runs
• G/C at 3' end helps binding