- The overhang sequence created is not dictated by the REase, and therefore no scar sequence is introduced.
- The fragment-specific sequence of the overhangs allows orderly assembly of multiple fragments simultaneously.
- The restriction site is eliminated from the ligated product, so digestion and ligation can be carried out simultaneously.
The net result is the ordered and seamless assembly of DNA fragments in one reaction. The accuracy of the assembly is dependent on the length of the overhang sequences. Therefore, Type IIS REases that create 4-base overhangs (such as BsaI/BsaI-HF®, BbsI/BbsI-HF, BsmBI and Esp3I) are preferred. Insert assembly calls for careful design of overhangs to direct the assembly, as well as verification that the Type IIS REase sites used are not present in the fragments for the assembly of the expected product. The use of web tools such as the NEB Golden Gate Assembly Tool greatly simplifies both processes, making Golden Gate Assembly a robust technology that assembles multiple DNA fragments (5), even if repetitive elements are present (6) and can, if wished, introduce multiple site-directed mutations (7). Golden Gate Assembly has been widely used in the construction of custom-specific TALENs for in vivo gene editing (8), among other applications.
NEB has developed an optimized mix for performing Golden Gate Assembly.
To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.
Visit Type IIS Restriction Enzymes for a comprehensive list of all Type IIS enzymes available from NEB and their characteristics.
- Engler, C., Kandzia, R., and Marillonnet, S. (2008) PLoS ONE 3, e3647.
- Engler, C., et al. (2009) PLoS ONE 4, e5553.
- Lee, J.H. et al, (1996) Genetic Analysis: Biomolecular Engineering, 13: 139-145.
- Padgett, K.A. and Sorge, J.A. (1996) Gene, 168: 31-35.
- Werner, S., et al. (2012) Bioeng. Bugs 3, 38-43.
- Scior, A., et al. (2011) BMC Biotechnol. 11, 87.
- Yan, P., et al. (2012) Anal. Biochem. 430, 65-67.
- Sanjana, N. E., et al. (2012) Nat. Protoc. 7, 171-192.
Protocols for Golden Gate Assembly
- A Typical Exonuclease V Reaction (M0345)
- Double Digest Protocol with Standard Restriction Enzymes
- Golden Gate Assembly Protocol for Using NEB Golden Gate Assembly Mix (E1600)
- Ligation Protocol with T4 DNA Ligase (M0202)
- Ligation Protocol with T7 DNA Ligase (M0318)
- Optimizing Restriction Endonuclease Reactions
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Quick Ligation Protocol (M2200)
- Recommended Screening Protocols for Using NEB Golden Gate Assembly Mix (E1600)
- Transformation Protocol
- Transformation Protocol for Using NEB Golden Gate Assembly Mix (E1600)
Molecular Cloning Technical Guide
The Molecular Cloning Technical Guide helps with product selection, protocols, tips for optimization and trouble-shooting.
Reagents and Tools for Molecular Cloning
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
- Restriction Endonucleases: Molecular Cloning and Beyond
- Synthetic Biology/DNA Assembly Selection Chart
- Type IIS Restriction Enzymes
- Troubleshooting Guide for Cloning
Other Tools & Resources
- Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T 2014. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system Sci Rep. 4, PubMedID: 24954249, DOI: 10.1038/srep05400
- 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), PubMedID: 24551083, DOI: 10.1371/journal.pone.0088218
- Feng Y, Zhang S, Huang X 2014. A robust TALENs system for highly efficient mammalian genome editing Sci Rep. 4, 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), PubMedID: 24581042, DOI: 10.1186/1754-1611-8-7
- Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez 2013. GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology Plant Physiology. 162(3), PubMedID: 23669743, DOI:
Publications related to Golden Gate Assembly
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Find out how Golden Gate Assembly can be used to quickly join multiple DNA fragments.
These days, restriction enzymes are being used in many more applications other than cloning. Learn more in this episode of NEB TV.
Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for reassembly. View a list of TypeIIS enzymes