Gibson Assembly

Gibson Assembly, also known as enzymatic or chew-back assembly, is a powerful tool used for DNA cloning. It allows you to manipulate almost any fragment of DNA in any location, alleviating the need to plan projects around restriction sites.

Overview and Background Info

Gibson Assembly uses sequence homology between fragments of DNA to join them together. A Gibson Assembly reaction mixture uses 3 enzymes: 5'-->3' exonuclease, high-fidelity polymerase and ligase. Together, these enzymes chew back the 5' end of DNA fragments (allowing fragments with sequence homology to anneal), fill in any gaps created by the chew-back, and seal the nicks in the annealed fragment. While it can be used to generate linear assemblies, Gibson is most commonly used to manipulate circular plasmids, for transformation and propagation in bacteria. Fragments for Gibson Assembly must have 15-500 bp of homology. This can be done through synthesis of entire fragments, or by amplification with "overhanging" primers

Protocol

Reaction

If using NEBuilder HiFi:

• Add 10 μL of DNA (+ water) to 10μL of 2xNEBuilder HiFi

If using homemade Master Mix:

• Add 5 or 10 μL of DNA (+ water, depending on indicated concentration of Master Mix) directly to tube containing 15 μL aliquot of master mix

Incubate at 50˚C for 45-75 mins (if Master Mix contains T7 DNA Ligase: incubate for additional 15-30 mins at 25˚C or room temp).
Transform 1-4 μL into competent bacteria, or store mixture at -20˚C.