Primer Design
Design primers for PCR amplification, Gibson Assembly, and Golden Gate cloning with built-in thermodynamic validation.
Supported methods
PCR primers
Standard PCR primer design. Configure melting temperature (Tm), GC content, primer length, and product size range. Nuclex returns ranked primer pairs with penalty scores.
| Parameter | Default | Range |
|---|---|---|
| Optimal Tm | 60°C | 50–72°C |
| Primer length | 20 nt | 18–30 nt |
| GC content | 40–60% | 20–80% |
| Product size | 200–1000 bp | 100–10000 bp |
Gibson Assembly primers
Generates primers with 20–40 bp overlapping tails for isothermal assembly. Binding regions are Tm-adjusted to ensure reliable amplification. Supports circular assemblies.
Golden Gate primers
Generates primers with Type IIS restriction enzyme recognition sites (BsaI or BbsI) and custom 4 bp overhangs. Warns if the target sequence contains internal recognition sites that could interfere with assembly.
Validation
Every primer is checked for:
- Hairpin formation — secondary structure ΔG threshold
- Homodimer — self-complementarity check
- Heterodimer — cross-primer complementarity (forward vs. reverse)
Validation uses thermodynamic modeling with nearest-neighbor parameters.
Export
Export primer results as CSV in IDT, Twist, or generic format. Each export includes primer name, sequence, Tm, GC%, and validation status.
Saving results
Primer designs are saved per-design and can be reloaded later. Each design stores the most recent primer result for each method (PCR, Gibson, Golden Gate).