Aug 28, 2024
Recombining DNA by Gibson Assembly
- 1University of Kent
Protocol Citation: Tobias von der Haar 2024. Recombining DNA by Gibson Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.261gee4yg479/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: March 20, 2017
Last Modified: August 28, 2024
Protocol Integer ID: 5278
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Abstract
Our protocol for producing recombinant DNA via Gibson Assembly, based on a home-made Gibson Master Mix.
Protocol materials
Dithiothreitol (DTT)MelfordCatalog # MB1015
Step 1
Deoxynucleotide Solution Set - 25 umol of eachNew England BiolabsCatalog #N0446S
Step 2
Taq DNA Ligase - 2,000 unitsNew England BiolabsCatalog #M0208S
Step 3
T5 Exonuclease - 1,000 unitsNew England BiolabsCatalog #M0363S
Step 3
Phusion DNA polymeraseNew England Biolabs
Step 3
Poly(ethylene glycol) 8000 [PEG 8000]Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510
Step 1
Tris BaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1503
Step 1
Poly(ethylene glycol) 8000 [PEG 8000]Sigma AldrichCatalog #89510
Tris BaseSigma AldrichCatalog #T1503
Dithiothreitol (DTT)MelfordCatalog # MB1015
Preparation of 5x Isothermal Buffer
Preparation of 5x Isothermal Buffer
Mix the following to give 1 ml of 5x Isothermal Buffer:
- 450 μl 50% PEG 8000
- 250 μl 2M Tris-HCl pH 7.5
- 100 μl 500 mM MgCl2
- 50 μl 1M DTT
- 100 μl 50 mM NAD
- 10 μl each of 100 mM ATP, CTP, GTP and TTP (PCR grade)
- 10 μl sterile water
Deoxynucleotide Solution Set - 25 umol of eachNew England BiolabsCatalog #N0446S
Preparation of Gibson Master Mix
Preparation of Gibson Master Mix
Mix the following reagents:
- 160 μl of 5x Isothermal Buffer
- 3.2 μl of 1U/μl T5 exonuclease (diluted 1:10 in water from the 10 U/μl stock)
- 10 μl of 2U/μl Phusion polymerase
- 80 μl of 40 U/μl Taq ligase
- 346.8 μl deionised water
Freeze in 15 μl aliquots (one 15 μl aliquot is ready-to-use for one Gibson assembly reaction)
Taq DNA Ligase - 2,000 unitsNew England BiolabsCatalog #M0208S
T5 Exonuclease - 1,000 unitsNew England BiolabsCatalog #M0363S
Phusion DNA polymeraseNew England Biolabs
Gibson Assembly
Gibson Assembly
Preparation: You need to have prepared two or more fragments of DNA which you wish to recombine, and which have 25-40 nucleotide homologous regions at their ends. For example, this could be a linearised vector and a PCR product which has sequences corresponding tot he ends of the linearised vector introduced via the primers.
- Mix the fragments to be assembled in equimolar ratios in a total volume of 5 μl. Add this mixture to one 15 μl aliquot of Gibson Master Mix.
- Incubate the reaction in a 50°C water bath or in a PCR machine at 50°C for one hour.
- Use 2-10 μl of the assembly reaction for a bacterial transformation.