NEBuilder® HiFi DNA Assembly

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.

4 Reasons to choose
NEBuilder HiFi 

  1. Save time – Enjoy simple and fast seamless cloning in as little as 15 minutes.
  2. Flexibility – Use one system for both "standard-size" cloning and larger gene assembly products, up to 6 fragments.
  3. Compatible with downstream applications – DNA can be used immediately for transformation or as template for PCR or RCA.
  4. Adaptable – Adapts easily for multiple DNA manipulations, including site-directed mutagenesis. 

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
  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.

  5. NEBuilder® HiFi DNA Assembly: Removal of 3´-end Mismatches

    Did you know that one of the advantages of NEBuilder HiFi is that it removes 3’ and 5’ -end mismatch sequences prior to fragment assembly? Learn how!

How does NEBuilder HiFi stack up?

graph iconPerformance data and advantages against NEB Gibson Assembly
graph iconPerformance data and advantages against In-Fusion HD
table iconSynthetic Biology/DNA Assembly Selection Chart
table iconComparison of DNA Assembly Reaction Types

Not your average DNA assembly reagent

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


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 


Protocols for NEBuilder® HiFi DNA Assembly

Learn More

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.