Primer Tm Calculator
Calculate DNA primer melting temperature using nearest-neighbour thermodynamics. Includes SantaLucia 1998 parameters with Na+ correction. Design primers for PCR, qPCR, and on-chip microfluidic reactions.
Parameters
Enter the primer sequence in 5' to 3' direction. Spaces and line breaks are ignored.
Typical PCR: 50 mM. On-chip reactions may vary.
Standard PCR: 250 nM. Microfluidic reactions: 50–500 nM.
Melting Temperature
| Property | Value |
|---|---|
| Length | 18 bp |
| GC Count | 6 |
| AT Count | 12 |
| GC % | 33.3 % |
| Molecular Weight | 6021.6 Da |
About Primer Melting Temperature
The melting temperature (Tm) is the temperature at which 50% of the DNA duplex is denatured. It is fundamental to PCR primer design, oligonucleotide synthesis, and hybridization-based assays. Accurate Tm calculation ensures specificity and efficiency of DNA amplification and detection.
Nearest-Neighbour Method (SantaLucia 1998)
This calculator uses the nearest-neighbour thermodynamic approach, which accounts for the sequence-dependent stability of each base pair. Unlike the simple formula (which depends only on GC content), nearest-neighbour calculations consider how each dinucleotide pair contributes to overall stability. The method includes correction for Na+ concentration (Owczarzy 2004), making it accurate across typical PCR and microfluidic conditions (10 mM to 1 M NaCl).
Use in Microfluidic PCR and On-Chip Reactions
On-chip PCR and qPCR in microfluidic devices benefit from precise Tm calculations. Microfluidic thermal cycling achieves faster heating and cooling than bulk reactions, but temperature gradients across the device may still affect annealing efficiency. Using accurate Tm values—adjusted for the specific Na+ and primer concentrations used in your chip—helps optimize primer sets and improve amplification uniformity. Many microfluidic platforms operate at lower primer concentrations (50–500 nM) than conventional PCR (1–2 µM).
Key Parameters
- GC Content: Higher GC% increases Tm. Optimal range is typically 40–60%.
- Primer Length: Longer primers have higher Tm. Typical PCR primers are 18–25 bp.
- Na+ Concentration: Higher salt increases Tm because it shields DNA negative charges. Most buffers contain 50–300 mM NaCl.
- Primer Concentration: Higher primer concentration increases Tm slightly (entropic effect).
Limitations
This calculator assumes fully complementary DNA duplexes without secondary structures. It does not account for hairpins, dimers, mismatches, or non-standard bases. Complex primer pools, formamide or DMSO additives, and non-aqueous solvents require manual adjustment. Always validate Tm experimentally before deploying primers in production or on-chip assays.
Design custom microfluidic PCR chips
From rapid prototyping in 3D-printed resin to production-scale injection moulding in COC and COP. Integrated thermal cycling and detection. Upload your design or get in touch.