Product Class: Kit

Gibson Assembly® Cloning Kit

Gibson Assembly Master Mix (NEB #M5510) has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10229799.

Have you tried NEBuilder HiFi DNA Assembly? NEBuilder HiFi offers several advantages over NEB Gibson Assembly. For more information, visit

Product Introduction

Gibson Assembly® allows for successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility.

  • Assembly and transformation in just under two hours
  • Flexible sequence design (scar-less cloning)
  • No PCR clean-up step required
  • High transformation efficiencies for inserts up to 20 kb
  • Easily adapted for multiple DNA manipulations, including site-directed mutagenesis
  • Includes competent cells
Catalog # Size Concentration
E5510S 10.0 reactions 2 X

Product Information


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 proprietary DNA 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.

Overview of the Gibson Assembly Cloning Method

Overview of the Gibson Assembly Cloning Method


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 on page 12. 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 Cloning Kit 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 and linearized vector using agarose gel electrophoresis, a NanoDrop™ instrument or other method.
  • Add fragments and linearized vector 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 NEB 5-alpha Competent E. coli (provided) or use directly in other applications.
This product is related to the following categories:
DNA Assembly, Cloning and Mutagenesis Kits Products
This product can be used in the following applications:
Gibson Assembly®

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Materials Required but not Supplied

DNA Polymerase (for generating PCR products):
We recommend Q5® High-Fidelity DNA Polymerase (NEB #M0491) or related products, such as Q5 Hot Start High-Fidelity DNA Polymerase (NEB #M0493) or Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494).
LB (Luria-Bertani) plates with appropriate antibiotic.


  • Assembly and transformation in just under two hours
  • Flexible sequence design (scar-less cloning)
  • No PCR clean-up step required
  • High transformation efficiencies for inserts up to 20 kb
  • Easily adapted for multiple DNA manipulations, including site-directed mutagenesis
  • Includes competent cells

Product Notes

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

  2. Storage Note:
    The kit is shipped on dry ice. Upon arrival, store kit at -80°C. After first use, store the kit components at indicated temperatures.

  3. Usage notes:

    To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following:

    • 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 at least 2–3 times higher than the concentration of vector. For assembly of multiple fragments into a vector, we recommend using equimolar ratio of fragments.
    • Transformation: NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) provided with the kit are recommended for use for assembled products of less than 20 kb in size. It is also possible to use other NEB competent E. coli strains, with the exception of BL21, BL21(DE3), Lemo21(DE3) and Nico21(DE3). For example, Shuffle T7 Express Competent E. coli can be used for the expression of a difficult to express protein. When using competent E. coli from a vendor other than NEB, we have seen decreased robustness of transformation with the Gibson Assembly reaction.
    • 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. Should you require the use of Electrocompetent cells, please use the Electrocompetent Cells Transformation Protocol.


  1. Gibson, D.G. (2009). Nature Methods. 343-345.
  2. Gibson, D.G. et al. (2010). Nature Methods. 901-903.
  3. Barnes, W.M. (1994). Proc. Natl. Acad. Sci.. 91, 2216-220.

Protocols, Manuals & Usage


  1. Gibson Assembly® Protocol (E5510)
  2. Gibson Assembly® Chemical Transformation Protocol (E5510)
  3. Gibson Assembly® Electrocompetent Cells Transformation Protocol (E5510)


The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting


  1. I am not sure whether to choose NEBuilder HiFi DNA Assembly or NEB Gibson Assembly? How are the products different?
  2. What are the advantages of this method compared to traditional cloning methods?
  3. How large of a DNA fragment can I assemble?
  4. How many fragments of DNA can be assembled in one reaction?
  5. What are the shortest overlaps that can be used with this assembly method?
  6. What are the longest overlaps that can be used with this method?
  7. Can ≤ 200 bp dsDNA fragments be assembled by this method?
  8. Can ssDNA oligonucleotides be combined and assembled with dsDNA fragments?
  9. Will the reaction work at other temperatures?
  10. Is it necessary to purify PCR products when doing DNA assemblies using either NEBuilder HiFi DNA Assembly Master Mix or Gibson Assembly Master Mix?
  11. Is it necessary to inactivate restriction enzymes after vector digestion?
  12. I would like to produce overlapping dsDNA fragments by PCR. Do I need to use PCR primers that have been purified by PAGE or HPLC?
  13. Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)?
  14. I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC?
  15. Can I PCR-amplify the assembled product?
  16. What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?
  17. How can I reduce the number of vector-only background colonies?
  18. What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly?
  19. Can I use electroporation instead of chemical transformation?
  20. Are there any differences between the Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Master Mix included in the Gibson Assembly Cloning Kit (NEB #E5510)?
  21. Are there any differences between the requirements for 2-3 fragmentassemblies versus 4–6?
  22. The Gibson Assembly Master Mix control reaction is not giving me any colonies. Why?
  23. When using a polymerase that doesn't contain a 3'-5' exonuclease activity (such as Taq DNA Polymerase) to amplify fragments to be used in a Gibson Assembly reaction, should I be concerned about the potential 3' mismatch generated by the addition of a non-templated nucleotide?
  24. When my Gibson Assembly Cloning Kit arrived, it was stored at -80°C. Will this harm the Gibson Assembly Master Mix?
  25. I would like to use NEBuilder but am concerned about user data privacy. How does NEB handle the information that I enter into NEBuilder?
  26. Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly?
  27. What antibiotic resistance is encoded in the NEBuilder Positive Control (assembly control)?

Quality, Safety & Legal

Quality Assurance Statement

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.

Specification Change Notifications

Gibson Assembly Master Mix (NEB #M5510) has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10229799. All subsequent (higher number) lots will contain rAlbumin.


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 DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

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 [email protected].

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.