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DNA Assembly and Cloning

DNA Assembly and Cloning

DNA assembly refers to a molecular cloning method that physically links together multiple fragments of DNA, in an end-to-end fashion, to achieve a designed, higher-order assembly prior to joining to a vector. This process is the cornerstone of the synthetic biology field and allows the construction of novel biological systems and devices using defined components. These techniques are carried out in vitro and are typically enzymatically driven with the final constructions being maintained in microbial host cells.

NEBuilder^® HiFi DNA Assembly and NEBridge^® Golden Gate Assembly products are the next-generation of tools for seamless cloning, assembly, and the synthetic biology field. These DNA assembly methods are optimized for quality, speed, flexibility, accuracy, scalability, and the ability to automate.

Which Assembly Method Should I Choose?

To help select the best DNA assembly method for your needs, please use the Product Summary Chart below and our Synthetic Biology/DNA Assembly Selection Chart for more details. If you are new to molecular cloning, you can review the advantages and disadvantages of each method by visiting the Which Molecular Cloning Technique Is Best For You page.


	NEBuilder^® HiFi DNA Assembly Learn More	NEBridge^® Golden Gate Assembly Learn More
STEPS IN PROTOCOL	Single tube (one-pot), only one reaction step	Single tube (one-pot), only one reaction step
CLONING EFFICIENCY	>95%	>95%
FRAGMENT SIZE	<100 bp to >10kb⁽¹⁾	<50 bp to >10 kb⁽²⁾
REACTION TIME(S)	From 15 minutes	From 5 minutes
FRAGMENT NUMBER	Up to 12	Up to 50+⁽³⁾
FORMATS	Available as Master Mix or full kit with chemically competent cells	Available as Master Mix or kits with optimized destination plasmid
IDEAL APPLICATION	Single insert cloning to medium complexity assemblies of 2-6 fragments; single-stranded oligo(s) to dsDNA bridge assembly; single or multi-site mutagenesis	Single insert cloning to highly complex assemblies of up to 30 fragments; sequences with high GC content and areas of repeats; dsDNA fragments < 100 bp
PRIMER DESIGN	15-30 bp overlaps for all fragment lengths and number of fragments. Supported by online primer design tool.	3-4 bp⁽⁴⁾ unique overhangs/overlaps with Type IIS recognition site. Supported by online primer design and ligase fidelity tools.

(1) The minimum recommended size for assembly of dsDNA fragments is 120 bp. For <100 bp fragments, single-stranded DNA oligos can be used.
(2) 50 bp to 10 kb have been validated internally. Sizes outside this range are possible.
(3) 50+ fragment assemblies require significant optimization. See Pryor et al (2022). Up to 30 fragments are recommended for routine assemblies and optimal performance.
(4) Overhang length depends on choice of Type IIS restriction enzyme.

Choose Type:

DNA Assembly and Cloning includes these areas of focus:

NEBuilder^® HiFi DNA Assembly
NEBridge^® Golden Gate Assembly
Gibson Assembly®
BioBrick® Assembly

Protocols for DNA Assembly and Cloning

Application Notes for DNA Assembly and Cloning

Tools & Resources

Feature Articles

Synthetic Genomics: Building a Better Bacterium

Restriction Endonucleases: Molecular Cloning and Beyond

Brochures

Molecular Cloning Technical Guide

Selection Tools

Synthetic Biology/DNA Assembly Selection Chart

Troubleshooting Guides

PCR Troubleshooting Guide

Troubleshooting Guide for Cloning

Troubleshooting Tips for Ligation Reactions

Usage Guidelines

Expanded “assembly standards” for MoClo, GoldenBraid2.0 and other modular Golden Gate Assembly methods

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

Technical Tips For Optimizing Golden Gate Assembly Reactions

Publications related to DNA Assembly and Cloning

Anton, B.P., Morgan, R.D., Ezraty, B., Manta, B., Barras, F., Berkmen, M. (2019) Complete genome sequence of Escherichia coli BE104, an MC4100 drivative lacking the methionine reductive pathway Microbiol Resour Announc; 8 (29), e00721-19. PubMedID: 31296691, DOI: 10.1128/MRA.00721-19
Potapov, V., Ong, J.L., Kucera, R.B., Langhorst, B.W., Bilotti, K., Pryor, J.M., Cantor, E.J., Canton, B., Knight, T.F., Evans, T.C., Lohman, G.J.S. (2018) Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly ACS Synth Biol; 7 (11), PubMedID: 30335370, DOI: 10.1021/acssynbio.8b00333
Feng Y, Zhang S, Huang X (2014) A robust TALENs system for highly efficient mammalian genome editing Sci Rep; 4, 3632. PubMedID: 24407151, DOI: 10.1038/srep03632
Binder A, Lambert J, Morbitzer R, Popp C, Ott T, Lahaye T, Parniske M (2014) A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants PLoS One; 9(2), e88218. PubMedID: 24551083, DOI: 10.1371/journal.pone.0088218
Abil Z, Denard CA, Zhao H (2014) Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA J Biol Eng; 8(1), 7. PubMedID: 24581042, DOI: 10.1186/1754-1611-8-7

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

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®.
Golden Gate Assembly Workflow

Find out how Golden Gate Assembly can be used to quickly join multiple DNA fragments.
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.
Listen to DAD (Data-optimized Assembly Design) when constructing high-complexity Golden Gate Assembly targets

This webinar introduces insights and modified protocols for Golden Gate Assembly enabling 50+ DNA fragments to be faithfully assembled in a single reaction.
NEBUILDER^® HIFI DNA ASSEMBLY^® : Bridging dsDNA with a ssDNA Oligo

Learn how NEBuilder® HiFi DNA Assembly bridges dsDNA with a ssDNA oligo.
Golden Gate Assembly Tool Tutorial

This video demonstrates how to use the Golden Gate Assembly Tool, we will walk through selecting insert and plasmids, primer design to make amplicon inserts.
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.
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.
Function and Application of Type IIS Restriction Enzymes

Learn about the functionality of Type IIS restriction enzymes and their role in molecular cloning, RNA therapeutic and vaccine development.