Cloning by Gibson Assembly
Eric ECS Cordeiro-Spinetti
Abstract
Gibson assembly is a molecular cloning method that allows for the joining of multiple DNA fragments in a single, isothermal reaction. It is named after its creator, Daniel G. Gibson, who is the chief technology officer and co-founder of the synthetic biology company, Telesis Bio. - Wikipedia
Daniel G. Gibson, of the J. Craig Venter Institute, described a robust exonuclease-based method to assemble DNA seamlessly and in the correct order, eponymously known as Gibson Assembly. The reaction is carried out under isothermal conditions using three enzymatic activities: a 5’ exonuclease generates long overhangs, a polymerase fills in the gaps of the annealed single strand regions, and a DNA ligase seals the nicks of the annealed and filled-in gaps. This method has been widely adopted and is a major workhorse of synthetic biology projects worldwide.
Steps
PCR (vector and insert)
Tm vector primers = oC
Tm insert primers = oC
Clean-up PCR products with 1µL Dpn1 for 0h 30m 0s at 37°C
Purify PCR products and resuspend in lowest volume possible (5-10 uL)
Set up Gibson ligation
Vector = 50-100ng
Molar ratio Vector/Insert = 1:1-3
Add to Gibson Master Mix
Incubate for 1h 0m 0sat 50°C
Transfer 1-2 µL into 50µL suspension of E.coli
Incubate on ice for 0h 30m 0s
Heat shock at40°C for 30 seconds
Transfer to 300µL of outgrowth media
Incubate in shaker for 1h 0m 0s at 37°C
Plate on antibiotic containing plate and grow 1h 0m 0s
Select colonies for sequencing
