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DNA Assembly and Cloning NEBuilder^® HiFi DNA Assembly

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

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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)

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

Online Supporting Tools

	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.
	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.

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FAQs for NEBuilder^® HiFi DNA Assembly

How does overlap length affect NEBuilder HiFi DNA Assembly efficiency?

Protocols for NEBuilder^® HiFi DNA Assembly

Application Notes for NEBuilder^® HiFi DNA Assembly

Tools & Resources

Web Tools

NEBuilder^® Protocol Calculator

Selection Tools

Synthetic Biology/DNA Assembly Selection Chart

Usage Guidelines

Optimization Tips for NEBuilder® HiFi DNA Assembly and NEB® Gibson Assembly

Legal Information

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.

Videos

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!
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.
NEBUILDER^® HiFi: the next generation of DNA Assembly

In this webinar, we will explore some of the latest applications for NEBuilder HiFi DNA Assembly. View full Q & A summary.
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^®.
Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly^® or Gibson Assembly^®

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.
NEBUILDER^® HIFI DNA ASSEMBLY^® : Bridging dsDNA with a ssDNA Oligo

Learn how NEBuilder® HiFi DNA Assembly bridges dsDNA with a ssDNA oligo.
NEBUILDER^® Assembly Tool 2.0 What’s New?

NEBuilder Assembly Tool can be used to design primers for your NEBuilder HiFi or Gibson Assembly reactions based on entered fragment sequences and the polymerase being used for amplification. This video will highlight some useful updates and the main differences between the two versions.
NEBUILDER^® Assembly Tool 2.0 Fragments Amplified by PCR

This video demonstrates how the NEBuilder Assembly Tool can be used to generate overlap sequences for the assembly of two fragments into a vector. In this scenario, all fragments are amplified by PCR and the vector does not have convenient restriction sites.
NEBUILDER^® Assembly Tool 2.0 Restriction Enzyme Digest

This video demonstrates how to use the NEBuilder® Assembly Tool to build a construct using a restriction enzyme digested vector and two PCR-generated inserts. The tool will help to design PCR primers containing the required overlap sequences. We will also regenerate one of the restriction enzyme recognition sites.
NEBioCalculator^® - Using the ds: mass < — > moles module to plan an NEBuilder^® HiFi DNA Assembly Reaction

This tutorial describes the use of the NEBioCalculator web tool module that converts mass to, or from, moles to help plan an NEBuilder HiFi DNA Assembly reaction