Gibson Assembly Reaction Calculator

How Gibson Assembly works

Gibson Assembly (Gibson et al., 2009) joins multiple DNA fragments with overlapping ends in a single isothermal reaction at 50°C. The reaction contains three enzymes:

1. T5 exonuclease — chews back 5′ ends, exposing complementary 3′ overhangs 2. Phusion DNA polymerase — fills in gaps 3. Taq DNA ligase — seals nicks

The overlapping ends anneal, and the polymerase/ligase repair the junctions to produce a seamless, scar-free assembly.

Molar calculations for DNA fragments

The molecular weight of dsDNA is approximately 650 Da per base pair. To calculate pmol:

NEB recommends 0.02–0.5 pmol per fragment for Gibson Assembly. For a typical 2-fragment assembly: 0.1 pmol each. For 4+ fragments: 0.05 pmol each to keep total volume within the reaction.

Designing overlaps for Gibson Assembly

Each fragment must share 15–40 bp of overlap with its neighbor. Key guidelines:

• Standard Gibson: 20–40 bp overlaps recommended
• NEBuilder HiFi: 15–25 bp overlaps sufficient (higher fidelity enzyme)
• Overlap Tm should be ≥48°C for efficient annealing at 50°C
• Avoid secondary structures, repeats, or high GC content in overlap regions
• Use a primer design tool to add overlaps via PCR or order overlapping gBlocks/synthetic fragments