Gibson Assembly Calculator

Calculate optimal fragment ratios and design overlaps for seamless Gibson assembly. Support for 2-15 DNA fragments with automatic molar ratio calculations.

Standard Gibson Assembly (Vector + Insert)

Linearized vector backbone
DNA fragment to insert
Recommended: 50 ng
Recommended: 2-3× for Gibson
Optimal: 15-40 bp (20-25 bp recommended)

Multiple Fragment Assembly

Ratio for each insert relative to vector
Insert Fragment 1

Overlap Design & Tm Calculator

Enter the overlap sequence to analyze

What is Gibson Assembly?

Gibson assembly is a molecular cloning technique for seamless assembly of multiple DNA fragments in a single isothermal reaction. It uses exonuclease, polymerase, and ligase enzymes to join DNA fragments with overlapping ends.

How to Use This Calculator

Quick guide for Gibson assembly:

  1. Enter vector and insert sizes
  2. Set vector amount (typically 50 ng)
  3. Choose insert:vector ratio (2-3× recommended)
  4. Design overlaps (20-25 bp optimal)
  5. View calculated DNA amounts
  6. Follow assembly protocol

When to Use Gibson Assembly

Ideal applications:

  • Seamless cloning without restriction sites
  • Multi-fragment assembly (2-15 fragments)
  • Site-directed mutagenesis
  • Scarless gene deletions
  • Synthetic biology constructs
  • Fusion protein creation

Advantages Over Traditional Cloning

Why choose Gibson assembly:

  • No restriction enzyme requirements
  • Seamless joining (no scars)
  • Assemble multiple fragments at once
  • High efficiency (typically >90%)
  • Single-step, isothermal reaction
  • Flexible fragment sizes (150 bp to 15 kb)

Optimal Parameters

Best practices for success:

  • Overlap length: 20-25 bp (range: 15-40 bp)
  • GC content: 40-60% in overlaps
  • Insert ratio: 2-3× molar excess
  • Total DNA: 50-100 ng recommended
  • Fragment number: 2-5 optimal, up to 15 possible
  • Incubation: 50°C for 15-60 min

Troubleshooting

Q: No colonies or low efficiency?
A: Check overlap design (length, GC%, Tm), use 2-3× insert excess, ensure fragments are gel-purified, verify PCR product quality.

Q: High background clones?
A: Use fresh master mix, ensure vector is linearized, avoid excess DNA (>200 ng total), include no-insert control.

Design Tips

Maximize assembly success:

  • Use high-fidelity PCR for overlap creation
  • Gel-purify all DNA fragments
  • Keep overlaps unique (no repeats)
  • Avoid strong secondary structures
  • DpnI digest template DNA after PCR
  • Test overlap quality with this calculator

FAQ

Q: Can I assemble >5 fragments?
A: Yes, but efficiency decreases. For >5 fragments, consider hierarchical assembly (assemble subsets, then combine).

Q: Do overlaps need to be identical?
A: Yes! The overlapping regions on adjacent fragments must be perfectly complementary sequences.