NEBuilder® HiFi DNA Assembly
Assembling DNA fragments is a key part of both synthetic biology techniques and cloning. NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA fragments, even those with 5´- and 3´-end mismatches. This flexible mix enables simple and fast seamless cloning utilizing a proprietary high-fidelity polymerase.
With over 9,500 citations, NEBuilder HiFi is the #1 cited product for DNA assembly. Find out why it is the most trusted choice for DNA assembly and cloning.
6 Reasons why researchers overwhelmingly choose NEBuilder HiFi
- Performance: Increase success and screen less with virtually error-free, high-efficiency cloning
- Speed: Complete the simple and fast seamless cloning protocol in as little as 15 min
- Flexibility: Easily accomplish both routine cloning and complex assemblies of 2 ‑ 12 fragments
- Compatibility: Use the DNA immediately for transformation or as a template for PCR or RCA
- Adaptability: Use single stranded DNA oligos, remove unwanted sequences, and perform multi-site directed mutagenesis
- Support: Increase success with free online tools for primer design and reaction calculations

Unique capabilities not offered by other DNA assembly methods
How does NEBuilder HiFi stack up?
Performance data and advantages against GeneArt® Gibson Assembly® and In-Fusion® Snap Assembly
Synthetic Biology/DNA Assembly Selection Chart
Comparison of DNA Assembly Reaction Types
Guidelines for using NEBuilder HiFi DNA Assembly
High-throughput cloning and automation solutions
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)
- 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
- Does not include competent cells
Online Supporting Tools
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NEBuilder® Assembly Tool Use to easily design primers for your NEBuilder HiFi DNA Assembly reactions. |
NEBuilder® Protocol Calculator Use to generate a customized protocol for your NEBuilder HiFi DNA Assembly reaction. |
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NEBaseChanger® For multi-site mutagenesis primer design using NEBuilder HiFi DNA Assembly. |
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 busdev@neb.com. The use of these products may require you to obtain additional third party intellectual property rights for certain applications.
Choose Type:
- NEBuilder® HiFi DNA Assembly Reaction Protocol (NEB #E2621, #E5520, #E2623)
- NEBuilder® HiFi DNA Assembly Electrocompetent Transformation Protocol
- NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol (E2621, E5520, E2623)
- NEBuilder Assembly of a PCR Fragment
- Protocol for Cloning Repeat Elements and Unstable DNA Inserts with NEBuilder® (NEB #C3040, #E5520, #E2621, #E2623)
- Protocol for Bridging double-stranded DNA with a single-stranded DNA oligo using NEBuilder HiFi DNA Assembly (NEB #E2621)
- Protocol for assembling annealed DNA oligonucleotides and a double-stranded DNA vector using NEBuilder HiFi DNA Assembly (NEB #E2621)
- Bridging dsDNA with a ssDNA Oligo and NEBuilder® HiFi DNA Assembly to create an sgRNA-Cas9 Expression Vector
- Improved methods for site-directed mutagenesis using NEBuilder® HiFi DNA Assembly Master Mix
- Improved method for assembly of linear yeast expression cassettes using NEBuilder® HiFi DNA Assembly Master Mix
- Construction of an sgRNA-Cas9 expression vector via single-stranded DNA oligo bridging of double-stranded DNA fragments
- NEBuilder® Protocol Calculator
- Synthetic Biology/DNA Assembly Selection Chart
- Optimization Tips for NEBuilder® HiFi DNA Assembly and NEB® Gibson Assembly
Web Tools
Selection Tools
Usage Guidelines
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. All other trademarks are the property of their respective owners. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.