Annealing Temperature Calculator
Calculate primer melting temperature (Tₘ) and annealing temperature (Tₐ) using basic and nearest-neighbor thermodynamic models with batch analysis and visualization tools.
Formulas Used
- Basic Rule:
- Tₘ = 4(G + C) + 2(A + T)
- Nearest Neighbor:
- Tₘ = (ΔH / (ΔS + R ln[Na⁺])) − 273.15 + 16.6 log₁₀[Na⁺]
- Annealing Temperature:
- Tₐ = Tₘ − offset (≈ 3–5°C)
- GC Content:
- GC% = (G + C) / Length × 100
PCR & Primer Analysis Details
- - Annealing temperature (Tₐ) determines primer binding specificity in PCR.
- - Too low Tₐ → non-specific binding.
- - Too high Tₐ → poor amplification.
- Melting Temperature (Tₘ):
- - Temperature where 50% of DNA duplex dissociates.
- - Depends on GC content, length, and salt concentration.
- Primer Pair Compatibility:
- - Forward & reverse primers should have similar Tₐ.
- - Difference ≤ 5°C is considered optimal.
- Batch Processing:
- - Supports multiple primer pairs via CSV or text input.
- - Automatically calculates statistics (mean, median, std).
- Sensitivity Analysis:
- - Evaluates effect of salt variation on Tₐ.
- - Helps optimize PCR conditions.
- Visualization:
- - GC vs AT distribution shown via color-coded diagram.
- - Bar chart compares annealing temperatures.
- Notes:
- - Valid DNA bases only: A, T, G, C.
- - Recommended primer length: 18–30 bases.
- - Salt concentration affects stability significantly.