Gibson Assembly® Master Mix

Catalog #	Concentration	Size	List Price	Quantity	Your Price
E2611L		50 reactions	$1,031.00		$927.90
E2611S		10 reactions	$261.00		$234.90

Catalog #	Size	List Price	Your Price
E2611L	50 reactions	$1,031.00	$927.90
E2611S	10 reactions	$261.00	$234.90

Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc. It allows for successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility. It has been rapidly adopted by the synthetic biology community due to its ease-of-use, flexibility and suitability for large DNA constructs.

Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction (1,2). The Gibson Assembly Master Mix includes three different enzymatic activities that perform in a single buffer:

The exonuclease creates single-stranded 3´ overhangs that facilitate the annealing of fragments that share complementarity at one end (overlap region).
The polymerase fills in gaps within each annealed fragment.
The DNA ligase seals nicks in the assembled DNA.

The end result is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation. The method has been successfully used by Gibson’s group and others to assemble oligonucleotides, DNA with varied overlaps (15–80 bp) and fragments hundreds of kilobases long (1–2).

To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.

For help designing primers, please view our primer design video.

Specification:

10 μl of 2X Gibson Assembly Master Mix was incubated with 6 fragments (5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmol each) in a final volume of 20 μl at 50°C for 60 minutes. NEB 5-alpha Competent E. coli (NEB #C2987) were transformed with 2 μl of the master mix/fragment mixture using the transformation protocol. Greater than 100 white colonies were observed when 1/10 of the outgrowth was spread on an ampicillin plate with IPTG/Xgal and incubated overnight.

Overview of Gibson Assembly Master Mix Protocol:

Design primers to amplify fragments (and/or vector) with appropriate overlaps
PCR amplify fragments using a high-fidelity DNA polymerase.
Prepare linearized vector by PCR amplification using a high-fidelity DNA polymerase or by restriction digestion.
Confirm and determine concentration of fragments using agarose gel electrophoresis, a Nanodrop™ instrument or other method
Add DNAs to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour, depending on number of fragments being assembled.
Transform into E. coli or use directly in other applications

Reagents Supplied

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

E2611S -20

	NEBuilder^® Positive Control	N2611AVIAL	-20	1 x 0.05 ml
	Gibson Assembly Master Mix	M5510AVIAL	-20	1 x 0.1 ml	2 X

E2611L -20

	NEBuilder^® Positive Control	N2611AVIAL	-20	1 x 0.05 ml
	Gibson Assembly Master Mix	M5510AAVIAL	-20	1 x 0.5 ml	2 X

Features

Increased number of successful assembly products, particularly for longer or greater number of fragments
Flexible sequence design (scarless cloning)
No clean-up step required
Complex assembly achieved in 1 hour
DNA can be used immediately for transformation, or as template for PCR or RCA
Easily adapted for multiple DNA manipulations, including site directed mutagenesis, insertions and deletions

Properties & Usage

Materials Required but not Supplied

DNA Polymerases (for generating PCR products):
We recommend Q5® High-Fidelity DNA Polymerase (NEB #M0491) or related products, such as Q5 Hot Start Flex DNA Polymerase (NEB #M0493), Q5 Hot Start Flex 2X Master Mix (NEB #M0494).

LB (Luria-Bertani) plates with appropriate antibiotic.

SOC Outgrowth Medium (NEB #B9020).

Competent Cells:
We recommend NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987). For assembled products greater than 10 kb, NEB recommends using NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) or NEB 10-beta Electrocompetent E. coli (NEB #C3020).

Storage Notes

Store at -20°C. Thaw, vortex thoroughly before use and keep on ice.

Companion Products

Notes

General notes:
We highly recommend using our web tool, NEBuilder™ to design PCR primers with overlapping sequences between the adjacent DNA fragments and for their assembly iinto a cloning vector.
Usage notes:

To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following:
- Cells: Transformation efficiency of competent cells can vary by several logs. Perceived assembly efficiency directly correlates to the competence of the cells used for transformation.
- Electroporation: Electroporation can increase transformation efficiency by several logs. When using the Gibson Assembly Master Mix product for electroporation, it is necessary to dilute the reaction 3-fold and use 1 μl for transformation.
- DNA: PCR product purification is not necessary if the total volume of all PCR products in the Gibson Assembly reaction is 20% or less of the Gibson Assembly reaction volume. Higher volumes of PCR products may reduce the efficiency of Gibson Assembly and transformation due to the elevated carryover amounts of PCR reaction buffer and unused primers present in the PCR product. Column purification of PCR products may increase the efficiency of both Gibson Assembly and transformation by 2–10 fold and is highly recommended when performing assemblies of three or more PCR fragments or assembling longer than 5 kb fragments. Purified DNA for assembly can be dissolved in ddH₂O (Milli-Q® water or equivalent is preferable), TE or other dilution buffers.
- Insert: When directly assembling fragments into a cloning vector, the concentration of assembly fragments should be 2–3 times higher than the concentration of vector. For assembly of 3 or more fragments, we recommend using equilmolar ratio of fragments.
- Biology: Some DNA structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.

References

Gibson, D.G. et.al (2009). NatureMethods. 343-345.
Gibson, D.G. et al. (2010). NatureMethods. 901-903.
Barnes, W.M. (1994). Proc. Natl. Acad. Sci.. 91, 2216-2220.

Protocols

FAQs

Citations

Additional Citations

Law SH, Sargent TD (2014) The Serine-Threonine Protein Kinase PAK4 Is Dispensable in Zebrafish: Identification of a Morpholino-Generated Pseudophenotype PLoS One 9(6), e100268.PubMedID: 24945275, DOI: 10.1371/journal.pone.0100268
Lipscomb GL, Schut GJ, Thorgersen MP, Nixon WJ, Kelly RM, Adams MW (2014) Engineering hydrogen gas production from formate in a hyperthermophile by heterologous production of an 18-subunit membrane-bound complex J Biol Chem 289(5), 2873-9.PubMedID: 24318960, DOI: 10.1074/jbc.M113.530725
Guilinger JP, Thompson DB, Liu DR (2014) Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification Nat Biotechnol 32(6), 577-82.PubMedID: 24770324, DOI: 10.1038/nbt.2909
Gai CS, Lu J, Brigham CJ, Bernardi AC, Sinskey AJ (2014) Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16 AMB Express 4(1), 2.PubMedID: 24410804, DOI: 10.1186/2191-0855-4-2
Vandergaast R, Hoover LI, Zheng K, Fredericksen BL (2014) Generation of West Nile virus infectious clones containing amino acid insertions between capsid and capsid anchor Viruses 6(4), 1637-53.PubMedID: 24721788, DOI: 10.3390/v6041637
Phelan VV, Moree WJ, Aguilar J, Cornett DS, Koumoutsi A, Noble SM, Pogliano K, Guerrero CA, Dorrestein PC (2014) Impact of a transposon insertion in phzF2 on the specialized metabolite production and interkingdom J Bacteriol 196(9), 1683-93.PubMedID: 24532776, DOI: 10.1128/JB.01258-13
Chinnici JL, Fu C, Caccamise LM, Arnold JW, Free SJ (2014) Neurospora crassa Female Development Requires the PACC and Other Signal Transduction Pathways, Transcription Factors, Chromatin Remodeling, Cell-To-Cell Fusion, and Autophagy PLoS One 9(10), e110603.PubMedID: 25333968, DOI: 10.1371/journal.pone.0110603

Quality Control Assay

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Change Notifications

Gibson Assembly Master Mix has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10229799. All subsequent (higher number) lots will contain rAlbumin. For additional information, please visit at NEB Restriction Enzyme formulations with Recombinant Albumin (rAlbumin).

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety Data Sheets

Legal And Disclaimer

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Limited Warranty: The Gibson Assembly® Master Mix and Gibson Assembly Cloning Kit are warranted to perform according to specifications stated on the certificate of analysis. No other warranty is made, whether express or implied, including any warranty of merchant ability or fitness for a particular purpose. This warranty limits NEB’s and its licensors’ liability to only the price of the product. Neither NEB nor its licensors shall have any responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever.

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