Sep 07, 2023

Public workspaceGibson Assembly Cloning

DOI
dx.doi.org/10.17504/protocols.io.eq2lyjwyqlx9/v1
  • 1Stanford University School of Medicine;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Suzanne R Pfeffer
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DOI: dx.doi.org/10.17504/protocols.io.eq2lyjwyqlx9/v1
Protocol Citation: Claire Y Chiang, Suzanne R Pfeffer 2023. Gibson Assembly Cloning . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjwyqlx9/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: September 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 87471
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's Disease
Grant ID: ASAP-000463
Abstract
Gibson assembly requires a vector backbone and one or more inserts that have been PCR amplified. The inserts should have 15-20 base pairs of overlap at ligation sites, so primers used to amplify the inserts should contain a tail overhang with this homology. The vector backbone should be linear.

A Gibson reaction uses a master mix that can be homemade or commercially purchased. Add the appropriate amount of master mix, then your vector and insert at 2-3 fold molar excess for a 20 µl reaction. Exact ratios will need to be optimized based on the size of your vector and inserts and how many inserts you have. Incubate this reaction at 50°C for 60 minutes. Then transform this reaction into DH5 alpha (or competent cell of choice) and plate on LB agar with appropriate antibiotic.
Materials
PEG-8000                             
Tris-HCl pH 7.5             MgCl2                                
DTT                                   
dNTPs [NEB, N0447S]       
NAD                                      10U/µl T5 exonuclease [NEB, M0663S] 2U/µl Phusion polymerase [Thermo, F530S] 40U/µl Taq Ligase [NEB, M0208S] Sterile water

Competent cells (DH5α) [Thermo, 18258012]
PCR machine
LB, LB agar
antibiotic of choice
Prepare Gibson master mix
Prepare Gibson master mix
Make 5 ml of 5x reaction buffer
25% PEG-8000 1.25 g
500 mM Tris-HCl pH 7.5 2.5 ml
50 mM MgCl2 0.25 ml of 1M
50 mM DTT 0.25 ml of 1M
1 mM each dNTPs 0.05 ml each of 100mM
5 mM NAD 0.5 ml of 50 mM
Prepare master mix

Master Mix
320 µl 5X Reaction Buffer (above)
0.64 µl 10U/µl T5 exonuclease
20 µl 2U/µl Phusion
160 µl 40U/µl Taq Ligase
700 µl Water
Use master mix as 1.5x. Snapfreeze in Amount15 µL aliquots and store at Temperature-80 °C .

Gibson assembly
Gibson assembly
Prepare linearized vector backbone by restriction enzyme digestion or PCR amplification.
Prepare inserts by PCR amplification. Ensure that each insert has 15-20 base pairs of overlap with other inserts/vector at ligation sites, added by primer design.
Take a 15 ul aliquot of 1.5x Gibson master mix on ice. Add 50-100 ng of vector with 2-3 fold molar excess of PCR inserts, and sterile water if necessary, to make total reaction volume up to Amount20 µL l.

Mix well and let incubate in PCR machine for Duration01:00:00 at Temperature50 °C

1h
Plasmid preparation
Plasmid preparation
1h
1h
After incubation is complete, transform into competent cells (DH5α) and plate onto LB agar with appropriate antibiotic. Let this grow at Temperature37 °C DurationOvernight .

Pick a single colony to grow in 5 ml LB culture at Temperature37 °C DurationOvernight and purify plasmid to check sequencing.