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.

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NEBuilder® HiFi DNA Assembly

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NEBridge® Golden Gate Assembly

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Single tube (one-pot), only one reaction step

Single tube (one-pot), only one reaction step

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<100 bp to >10kb(1)

<50 bp to >10 kb(2)

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From 15 minutes

From 5 minutes

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Up to 12

Up to 50+(3)

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Available as Master Mix or full kit with chemically competent cells

Available as Master Mix or kits with optimized destination plasmid

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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

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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
    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
    • 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
    • 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
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. 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

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.


Need more help selecting
a product for DNA assembly?

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View our Synthetic Biology/DNA Assembly Selection Chart for more details.

View Selection Chart

Learn more about automation for cloning

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With automation, researchers can scale up and increase throughput, as well as save time and money with rapid workflows.

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Which molecular cloning
technique is best for you?

For the new cloner, NEB suggests choosing one of three cloning methods. Find the method that works for your application.

For the new cloner, NEB suggests choosing one of three cloning methods. Find the method that works for your application.

Find a Cloning Method