Feb 26, 2026

Public workspaceMake Gibson Assembly 2X mastermix

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvj1jrwvk5/v1
  • Goosang Yu1
  • 1New York Genome Center, New York, USA
Goosang Yu
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvj1jrwvk5/v1
Protocol Citation: Goosang Yu 2026. Make Gibson Assembly 2X mastermix. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj1jrwvk5/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: February 13, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 243115
Keywords: Gibson assembly, Molecular cloning, homemade, nebuilder hifi mix, gibson assembly 2x, gibson assembly, homemade gibson mix, times cheaper than the nebuilder hifi mix, traditional t4 ligation, ssdna from endonuclease activity, ssdna, efficient cloning, based cloning method, cloning method
Abstract
Gibson assembly, a homology-based cloning method, enables much simpler and more efficient cloning compared to traditional T4 ligation. The commercially available product based on this method is the NEBuilder‱ HiFi DNA Assembly Master Mix (NEB, E2621), which has also been widely used in many labs. This protocol follows the method refined by Rabe et al. (2020), and is directly based on the procedure summarized by Julian Wills from the David Liu lab. The key point of this protocol is the addition of SSB to protect ssDNA from endonuclease activity.

The homemade Gibson mix is about 6.5 times cheaper than the NEBuilder HiFi mix.



Guidelines
It is a very simple procedure that only requires thoroughly mixing the prepared components. All mixture preparations should be performed in ice-cold tubes. Since the 50% PEG8000 solution is highly viscous, it should be pipetted very slowly and carefully. For the enzymes, it is best to use ones with sufficient remaining shelf life (well before their expiration date).
Materials
Reagent
  • Reagent1M Tris pH 7.5 Merck MilliporeSigma (Sigma-Aldrich)
  • Reagent1M MgCl2 solutionThermo Fisher ScientificCatalog #AM9530G
  • ReagentPoly(ethylene glycol) 8000 [PEG 8000]Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510
  • Reagentbeta-Nicotinamide adenine dinucleotide (NAD+) - 0.2 mlNew England BiolabsCatalog #B9007S
  • Reagent1M DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816
  • ReagentDeoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
  • ReagentPurified Water for MicrobiologyThermo FisherCatalog #BO0184B

Enzymes
  • ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
  • ReagentT5 Exonuclease - 1,000 unitsNew England BiolabsCatalog #M0363S
  • ReagentET SSB - 50 ugNew England BiolabsCatalog #M2401S
  • ReagentTaq DNA LigaseMCLABCatalog #TDL-200









Troubleshooting
Problem
PEG 8000 does not dissolve easily when preparing the solution.
Solution
Heat it at 60–70°C for about 5–10 minutes, or microwave it for approximately 20–30 seconds.
Before start
The manufacturer of the enzymes used is generally not critically important. We primarily use products from NEB, but for Taq DNA ligase, which accounts for a significant portion of the cost, we chose a more affordable alternative supplier. If you plan to prepare a master mix using enzymes from different manufacturers, I recommend first testing with small quantities before scaling up for large-volume use.
The original Gibson Assembly master mix used Phusion DNA polymerase, but in this protocol, we used Q5 polymerase instead. There was no specific technical reason for this change—our lab does not commonly use Phusion, and we have ample stock of Q5. In general, using a different polymerase should not cause major issues, but if you decide to switch polymerases, I strongly recommend validating it first on a small scale. The original paper recommends using a polymerase with proofreading activity.

This protocol follows a modified approach that includes the addition of an SSB (single-strand binding domain). This modification has been reported to improve cloning efficiency when assembling multiple inserts, and in my experience, it also works very well for library construction using oligo pools. Because of the presence of SSB, it may be possible to incorporate single-stranded DNA without PCR amplification, although I have not experimentally validated this.
Make Gibson assembly 2X mixture
Mix following reagents to make 5X isothermal reaction buffer.
ReagentVolume5X Conc.Final Conc. (1x)
1M Tris-HCl, pH 7.51mL500mM100mM
1M MgCl2100uL50mM10mM
10mM dNTP mix200uL1mM200uM
1M DTT (dithiothreitol)100uL50mM10mM
50mM NAD+ (10X) [NEB B9007S]200uL5mM1mM
Purified water400uL--
Total2mL
Mix following enzymes with 5X isothermal reaction buffer.
* For a 50% w/v solution, dissolve 2.5g of PEG 8000 in hot water (80c, macrowaved water) or buffer and adjust the volume to 5 mL.
ReagentVolume2X Conc. or UnitsFinal Conc. (1x)
Q5 High-Fidelity DNA Polymerase20uL40 units
T5 Exonuclease1.2uL12 units
Taq DNA Ligase160uL6,400 units
ET SSB10uL--
5x Isothermal Reaction Mix320uL--
PEG 8000 (50% w/v)160uL10% w/v5% w/v-
Purified water129uL--
Total800uL
Molecular cloning by Gibson AssemblyUntitled section
When designing the Gibson assembly, I used the following conditions:
  • Backbone vector: 50 ng per reaction
  • Vector:Insert ratio: 1:5 (<1 kb), 1:3 (general), 1:2 (>5 kb)
  • Homology arms: ~25 nt for each fragment (range to 20-40nt)

DNA mix: Cut vector 50ng + Fragments (1:3 mol.ratio each) = 5uL
2X Gibson mix = 5uL
Total 10uL reaction
* even smaller amount reaction works very well (e.g. total 4uL (2uL DNA mix + 2uL Gibson mix
Incubate 50c 30min (1hr for 3 fragments or more)
Transformation:
  • mix 5uL of gibson product with 20uL of Stbl Competent cells,
  • incubate 4c 15min
  • heat shock 42c 80sec
  • Chill on 4c 1min
  • (Not required for Amp plate) Outgrowth in 50uL SOC at 37C with shaking
  • Plate on Amp LB plate




Protocol references
Rabe, Brian A., and Constance Cepko. "A simple enhancement for Gibson isothermal assembly." BioRxiv (2020): 2020-06.