Gibson & HiFi Assembly Planner
Homology arm designer for Gibson and NEBuilder HiFi assembly with SantaLucia nearest-neighbour Tm matching.
How it works
Assembly primers are designed by extending each fragment with homology to its neighbour. Overlap length is chosen to reach a target melting temperature calculated with nearest-neighbour thermodynamics, so that all junctions anneal under the same conditions during the isothermal assembly reaction.
Frequently asked questions
How long should Gibson assembly overlaps be?
Overlaps of 20–40 base pairs work for most assemblies, with 25–30 bp a common default. What matters more than length is that the overlap has a melting temperature at or above the assembly temperature — typically 48–50 °C for a 50 °C reaction — and that GC-rich junctions are kept shorter while AT-rich junctions are extended to reach the same Tm.
Why did my Gibson assembly fail?
The most frequent causes are insufficient or mismatched homology at a junction, unequal fragment molar ratios, and carry-over of uncut vector producing a background of empty colonies. Verify fragment sizes on a gel before assembly, use roughly equimolar amounts with a slight insert excess, treat linearised vector with phosphatase or DpnI to remove template, and confirm each junction sequence matches exactly.
Method reference
Gibson DG et al. (2009) Nat Methods 6(5):343-345.