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.

5 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. 
  5. Save money - No licensing fees from NEB for NEBuilder products

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

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 



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.

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Protocols for NEBuilder® HiFi DNA Assembly

Application Notes NEBuilder® HiFi DNA Assembly