Jun 14, 2023

Public workspaceCloning by Gibson Assembly

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxmp8gx1/v1
  • 1University of Miami
Eric ECS Cordeiro-Spinetti
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzxmp8gx1/v1
Protocol Citation: Eric ECS Cordeiro-Spinetti 2023. Cloning by Gibson Assembly . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxmp8gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 14, 2023
Last Modified: June 14, 2023
Protocol Integer ID: 83426
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.
PCR (vector and insert)
Tm vector primers = oC
Tm insert primers = oC
Clean-up PCR products with Amount1 µL Dpn1 for Duration00:30:00 at Temperature37 °C

30m
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 Duration01:00:00 at Temperature50 °C

1h
Transfer 1-2 µL into 50µL suspension of E.coli
Incubate on ice for Duration00:30:00

30m
Heat shock atTemperature40 °C for 30 seconds

Transfer to Amount300 µL of outgrowth media

Incubate in shaker for Duration01:00:00 at Temperature37 °C

1h
Plate on antibiotic containing plate and grow DurationOvernight

1h
Select colonies for sequencing
Protocol references