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´ restriction enzyme mismatches. This flexible kit enables simple and fast Seamless Cloning utilizing a new proprietary high-fidelity polymerase. Find out why NEBuilder HiFi is the next generation of DNA assembly and cloning.

    Benefits over NEB Gibson Assembly® Master Mix

    • Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly
    • Join DNA fragments together more efficiently, even with larger fragments or low DNA inputs
    • Use NEBuilder HiFi in successive rounds of assembly, because it removes 5´- and 3´- restriction enzyme mismatches
    • Bridge two double-stranded fragments with a synthetic single-stranded DNA oligo for simple and fast construction (e.g. linker insertion or gRNA libraries)
    • Switch from other systems easily, because NEBuilder HiFi is compatible with Gibson Assembly-designed (and other) fragments
    • Save time with simple and fast Seamless Cloning
    • No licensing fee requirements from NEB for the NEBuilder products
    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 DNA Assembly kits are available in various formats: with NEB 5-alpha competent cells (Cloning Kit, NEB #E5520), as a bundle with NEB 10-beta competent cells (Bundle for Large Fragments, NEB #E2623) and without competent cells (Master Mix, NEB #E2621) . NEB 5-alpha competent cells cells are excellent for routine assemblies of 15kb or less. 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 15kb. If the assembled genes contain repetitive sequences, NEB Stable Competent E. coli (NEB #C3040) should be used. 

    NEBuilder has been used in various applications, including:
    Site-directed mutagenesis
    Construction of an sgRNA-Cas9 expression vector
    Assembly of linear yeast expression cassettes

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



    NEBuilder HiFi DNA Assembly Master Mix offers improved efficiency and accuracy over NEB Gibson Assembly with different numbers of fragments.

    Reactions were set up in a 2- and 6- fragment assembly reaction according to recommended reaction conditions.

    NEBuilder HiFi DNA Assembly Master Mix offers improved efficiency and accuracy over NEB Gibson Assembly, with lower amounts of DNA by increasing overlaps.

    Reactions were set up in a 4- fragment assembly reaction according to recommended reaction conditions. Amount of DNA and size of overlap is shown.

    NEBuilder HiFi DNA Master Mix offers improved fidelity over NEB Gibson Assembly Master Mix

    Fidelity of assembled products was compared between NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) and NEB Gibson Assembly Master Mix (NEB #E2611). Experiments were performed using various fragment and vector sizes following suggested protocols. Experiments D and E did not yield any colonies with NEB Gibson Assembly. Experiments H and J were performed with fragments containing 3´ restriction enzyme mismatches. Experiments B through G vary because sequences of fragments are different

    NEBuilder HiFi DNA Assembly Workflow




    Protocols for NEBuilder® HiFi DNA Assembly