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  • Gibson Assembly® Cloning

    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. The technique allows for the successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility.

    The Gibson Assembly Cloning Kit (NEB #E5510) combines the power of Gibson Assembly Master Mix (NEB #E2611) with NEB 5-alpha Competent E. coli (NEB # C2987), enabling fragment assembly and transformation in just under two hours.

    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 a single-stranded 3´ overhang that facilitates the annealing of fragments that share complementarity at one end.
    • The polymerase fills in gaps within each annealed fragment.
    • The DNA ligase seals nicks in the assembled DNA.

    The assembled, fully-sealed construct is then transformed into NEB 5-alpha Competent E. coli. The entire protocol, from assembly to transformation, takes a little over an hour.

    • Introduction to Gibson Assembly®

      Watch an interactive tutorial that details the process by which Gibson Assembly® joins DNA fragments in a single tube, isothermal reaction.

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    • Primer Design and Fragment Assembly Using Gibson Assembly®

      Watch an interactive tutorial on primer design to see how simple it really is to clone with the Gibson Assembly® Cloning Kit.

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    Speed up your experimental design with our primer design tool

    Gibson Assembly Workflow

    Gibson Assembly employs three enzymatic activities in a single-tube reaction: 5´ exonuclease, the 3´ extension activity of a DNA polymerase and DNA ligase activity. The 5´ exonuclease activity chews back the 5´ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. The NEB Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Cloning Kit (NEB #E5510S) enable rapid assembly at 50˚C.

    Benefits of the Gibson Assembly Master Mix:

    • Increased number of successful assembly products, including 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

    Additional Benefits of the Gibson Assembly Cloning Kit:

    • All the advantages of the Gibson Assembly Master Mix
    • Includes competent cells
    • High transformation efficiencies for inserts up to 20 kb
    • Assembly and transformation in just under two hours

    References

    1. Gibson, D.G. et al. (2009) Nature Methods, 343–345.
    2. Gibson, D.G. et al. (2010) Nature Methods, 901–903.

    Here is what Customers Are Saying About Gibson Assembly

    “It is very convenient and fast to clone a gene of interest into a large vector, in which unique restriction sites are difficult to find or manipulate.”
    Researcher, Washington University

    “The Gibson Master Mix is a superior cloning tool that inevitably saves large blocks of time, increases work flow, eliminates false positive clones, and saves a great deal of money! Compared to traditional cloning, the elimination of having to produce multiple intermediate clones for multiple fragment ligations is an enormous time saver. Furthermore, the fact that it works the first time, every time saves you from the frustrating and time consuming troubleshooting steps. I easily saved a week’s worth screening and subcloning with this mix. My approach to cloning anything is forever changed.”
    Researcher; Sanford-Burnham Medical Research Institute

    Some components of this product are manufactured by New England Biolabs, Inc. under license from Synthetic Genomics, Inc.


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    Benefits of the Gibson Assembly Cloning Kit

    • Assembly and transformation in a little over an hour
    • 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

    Gibson Assembly Cloning Kit Provides Robust Transformation Efficiencies

    Assembly reactions containing 25 ng of linear pUC19 vector and 0.04 pmol of each fragment were performed following individual suppliers’ recommended protocols and using the competent cells provided with the kit. The total number of recombinant colonies was calculated per 25 ng of linear pUC19 vector added to the assembly reaction.

    One or Two Fragment Cloning Can be Easily Achieved Using Gibson Assembly

    Reactions cloning one 3 kb PCR fragment (A) or two 1.5 kb PCR fragments (B) into either Ndel-NotI digested pET-21a or PCR-amplified pET21a vector were set up and transformed according to recommended reaction conditions found in the manual. Assembly efficiency was calculated as a percentage of blue colonies (lacZ gene insert) versus a total number of colonies.

    High-Efficiency Cloning Can be Achieved with Inserts Up to 10 kb

    Outgrowth was diluted 10-fold with fresh SOC medium and 50 μl of the diluted outgrowth was plated on the three LB + Amp + IPTG + X-gal plates. Assembly efficiency was calculated as a percentage of white colonies (recombinant) versus the total number of colonies.