Mar 19, 2025

Public workspaceGibson Assembly

DOI
https://dx.doi.org/10.17504/protocols.io.ewov1de6yvr2/v1
  • Patricia Yuste-Checa1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany
Patricia Yuste-Checa
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1de6yvr2/v1
Protocol Citation: Patricia Yuste-Checa, F Ulrich Hartl 2025. Gibson Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1de6yvr2/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 11, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124321
Keywords: ASAPCRN, dna plasmids by gibson assembly, gibson assembly this protocol detail, gibson assembly, dna plasmid, new england biolab, dna in the correct order, assembly, dna, e2621, exonuclease, gibson assembly this protocol
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to clone DNA plasmids by Gibson assembly (New England Biolabs, E2621), an exonuclease-based method to easily assemble DNA in the correct order.
Materials
  • ReagentWizard SV Gel and PCR Clean-Up SystemPromegaCatalog #A9281
  • Reagent NEBuilder HiFi DNA Assembly Master MixNew England BiolabsCatalog #E2621

Troubleshooting
Purification of DNA Fragments
Prepare the DNA fragments by digestion with restriction enzyme or PCR amplification.

Note
  • DNA fragments can be also synthesized by companies like Twist Bioscience.

  • Ensure that the DNA fragments have overlapping ends of 15-20 base pairs.

Purify the DNA fragments by agarose gel electrophoresis, followed by excision of the corresponding band and extracting the DNA from the gel piece using a clean-up system like the Wizard SV Gel and PCR Clean-Up System.
Determine the DNA concentrations with a Nanodrop spectrophotometer.
Gibson Assembly
1h
For a reaction with 2-3 fragments, prepare the following mixture TemperatureOn ice , Molar ratio vector:insert = 1:2

  • Amount50 ng of vector (or bigger DNA fragment) + double in pmol of insert(s) + Amount10 µL NEBuilder HiFi DNA Assembly Master Mix + to Amount20 µL deionized H2O.

Note
  • pmols = (weight in ng) x 1,000 / (base pairs x 650 Daltons)
  • For other number of fragments and other detailed information please refer to, https://www.neb.com/en/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
  • Other total reaction volume can be set as far as the Master Mix (2x) is diluted to a final concentration of 1x.

Pipetting
Mix
Temperature
Incubate at Temperature50 °C for Duration01:00:00 .

1h
Incubation
Temperature
Place the reaction TemperatureOn ice .

Temperature
Transform Amount50 µL of competent bacteria with Amount2 µL of the Gibson assembly reaction.

Note
Use of NEB Stable Competent E. coli (High Efficiency) increases the efficiency of the cloning.


Pipetting