Jun 14, 2023

Cloning by Gibson Assembly

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzxmp8gx1/v1
  • 1University of Miami
Eric ECS Cordeiro-Spinetti
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzxmp8gx1/v1
Protocol Citation: Eric 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
Keywords: molecular cloning method, synthetic biology project, joining of multiple dna fragment, dna ligase, major workhorse of synthetic biology project, gibson assembly, multiple dna fragment, synthetic biology company, polymerase, dna, telesis bio, enzymatic activity, exonuclease, robust exonuclease, single strand region, isothermal reaction, annealed single strand region
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 1 µL Dpn1 for 00:30:00 at 37 °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 01:00:00 at 50 °C

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

30m
Heat shock at40 °C for 30 seconds

Transfer to 300 µL of outgrowth media

Incubate in shaker for 01:00:00 at 37 °C

1h
Plate on antibiotic containing plate and grow Overnight

1h
Select colonies for sequencing
Protocol references