NEBuilder® HiFi DNA Assembly

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  • NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA fragments, even those with 5´- and 3´-end mismatches. This flexible kit enables simple and fast seamless cloning utilizing a proprietary high-fidelity polymerase. Find out why NEBuilder HiFi is the next generation of DNA assembly and cloning.

    NEBuilder HiFi DNA assembly offers unique features over other DNA assembly methods, including:

    • 3´- and 5´-end mismatch removal
    • Bridging of two ds-fragments with a ssDNA oligo
    • Improved methods for site-directed mutagenesis

    View performance data against NEB Gibson Assembly® and In-Fusion® HD. To help select the best DNA assembly method for your needs, please use our Synthetic Biology/ DNA Assembly Selection Chart.

    1. Construction of an sgRNA-Cas9 Expression Vector via an ssOligo Bridge

      Learn how you can use NEBuilder HiFi to generate an sgRNA-Cas9 expression vector with a single-stranded oligo bridge.

    2. Introduction to NEBuilder HiFi DNA Assembly

      Find out how NEBuilder® HiFi DNA Assembly can reliably join DNA fragments in a single tube, isothermal reaction, with advantages over NEB Gibson Assembly®.

    3. Primer Design and Fragment Assembly Using NEBuilder® HiFi DNA Assembly or Gibson Assembly® E5510 E5520

      Watch an interactive tutorial on primer design to see how simple it really is to clone with either NEBuilder HiFi DNA Assembly or the Gibson Assembly Cloning Kit.

    4. NEBuilder® HiFi DNA Assembly: Bridging dsDNA with a ssDNA Oligo

      Learn how NEBuilder® HiFi DNA Assembly bridges dsDNA with a ssDNA oligo.

    NEBuilder HiFi products

    NEBuilder HiFi DNA Assembly Cloning Kit (NEB #E5520)
    • Includes NEB 5-alpha competent cells
    • NEB recommends NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) for routine assemblies of 15 kb or less
    • For repetitive sequences, NEB recommends NEB Stable Competent E. coli (NEB #C3040)
    NEBuilder HiFi DNA Assembly Bundle for Large Fragments (NEB #E2623)
    • Includes NEB 10-beta competent cells
    • NEB recommends NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) or NEB 10-beta Electrocompetent E. coli (NEB #C3020) for assemblies larger than 15 kb
    NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621)
    • Does not include competent cells 



    Not your average DNA assembly reagent

    NEBuilder HiFi DNA Assembly can be used for a variety of DNA assembly methods.



    Protocols for NEBuilder® HiFi DNA Assembly

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    One or more of these products are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at The use of these products may require you to obtain additional third party intellectual property rights for certain applications. 

    GIBSON ASSEMBLY® is a registered trademark of Synthetic Genomics, Inc.

    IN-FUSION® is a registered trademark of ClonTech Laboratories, Inc.