DNA Melting Temperature (Tm) Calculator
Calculate DNA melting temperature using the Wallace rule: Tm = 2(A+T) + 4(G+C). For short oligonucleotides under standard salt conditions. Essential for PCR primer design and hybridization.
🧬 Molecular Biology📐 Tm = 2(A+T) + 4(G+C)🧬 Biology
Number of A+T bases
Number of G+C bases
Please enter valid values.
Formula & Reference
| Variable | Symbol | Formula | Units |
|---|---|---|---|
| DNA Melting Temperature (Tm) Calculator | — | Tm = 2(A+T) + 4(G+C) | degrees C |
Step-by-Step Examples
Example 1
PCR Primer Tm
Primer: 5'-ATGCGATCGGCTATCCGAT-3' (20-mer). A=4, T=4, G=7, C=5.
- A+T = 8, G+C = 12
- Tm = 2(8) + 4(12) = 16 + 48 = 64 degrees C
- Annealing temp: 64 - 5 = 59 degrees C
- For paired primers, use lower Tm minus 5 degrees C
✓ Tm = 64 degrees C, anneal at 59 degrees C
Example 2
Short Probe (15-mer)
AT-rich probe: A+T=11, G+C=4.
- Tm = 2(11) + 4(4) = 22 + 16 = 38 degrees C
- Short AT-rich probes melt at low temperatures
- Increase G+C content or length to raise Tm
✓ Tm = 38 degrees C
Example 3
GC-rich Primer (22-mer)
A+T=6, G+C=16.
- Tm = 2(6) + 4(16) = 12 + 64 = 76 degrees C
- Very high Tm — may cause secondary structures
- Consider redesigning to reduce GC content
✓ Tm = 76 degrees C
Real-World Applications
PCR Design
Primer Tm determines annealing temperature for PCR cycling conditions.
Hybridization Arrays
Probe Tm determines hybridization temperature for microarrays and blots.
RT-PCR
Consistent Tm across primer pairs important for multiplex PCR reactions.
Molecular Diagnostics
Probe Tm calibration critical for quantitative PCR (qPCR) assay design.
Common Mistakes to Avoid
⚠️
Using Wallace rule for long sequences
Wallace rule (Tm = 2AT + 4GC) is only valid for short oligos (<20 bp) in 50mM NaCl. For longer sequences, use nearest-neighbor method or SantaLucia tables.
⚠️
Setting annealing temperature too high
Annealing temperature is typically Tm - 5 degrees C. Too high: poor amplification. Too low: non-specific bands.
⚠️
Ignoring primer-dimer potential
Two primers with complementary 3' ends form primer-dimers. Check with OligoCalc or Primer3 even if Tm looks fine.
Frequently Asked Questions
What is the Wallace rule? ▾
Tm = 2(A+T) + 4(G+C). Simple empirical formula for short DNA duplexes. Based on G-C pairs having 3 hydrogen bonds (stronger) vs A-T pairs having 2 (weaker).
When should I use nearest-neighbor instead? ▾
For oligos > 20 bp or when precision matters (cloning, diagnostic assays). Nearest-neighbor accounts for base stacking interactions between adjacent bases.
What salt concentration does Wallace rule assume? ▾
50 mM NaCl, 50mM KCl. Higher salt stabilizes duplexes (higher Tm); lower salt destabilizes. For 1M NaCl, add 16.6 x log(1/0.05) ≈ +22 degrees C correction.
Why is GC content important? ▾
G-C pairs form 3 hydrogen bonds vs 2 for A-T. More GC = higher Tm. Optimal primers have 40-60% GC content.
What is annealing temperature in PCR? ▾
The temperature at which primers bind to template, typically Tm - 5 degrees C. Too low: nonspecific binding. Too high: primer doesn't bind. Often optimized experimentally with gradient PCR.