Golden Gate Assembly

With constant advances both in the development of new enzymes (e.g., PaqCI, BsaI-HFv2 and BsmBI-v2) and research on maximizing enzyme functionality (e.g., ligase fidelity), NEB has become the industry leader in pushing the limits of Golden Gate Assembly (and related methods such as MoClo, GoldenBraid, Mobius Assembly and Loop Assembly). NEB has all the products and information you need to perform complex assemblies, as demonstrated with 35+ fragment assemblies exhibiting high efficiencies and >90% accuracy, along with low backgrounds.

Golden Gate Assembly Workflow for complex assemblies

Advances in ligase fidelity

Research at NEB has led to an increased understanding of ligase fidelity, including the development of a comprehensive method for profiling end-joining ligation fidelity in order to predict which overhangs have improved fidelity (1, 2). Their research has enabled complex fragment assemblies with high efficiencies and >90% accuracy. Use our Ligase Fidelity Tools to design high fidelity Golden Gate Assemblies under various experimental conditions.

Type IIS restriction enzymes for Golden Gate Assembly

NEB offers more Type IIS (i.e., recognize asymmetric DNA sequences and cleave outside of their recognition sequence) restriction enzymes than any other supplier, many of which are used in Golden Gate Assembly. NEB is pleased to offer: Esp3I (an isoschizomer of BsmBI that is recommended for use at 37°C and is supplied with CutSmart^® Buffer), PaqCI (an AarI isoschizomer with a 7 basepair recognition sequence) and the improved BsaI-HFv2 and BsmBI-v2, which are optimized for Golden Gate Assembly.

For information about other Type IIS restriction enzymes used in Golden Gate Assembly, such as BbsI, BbsI-HF and SapI, view our Type IIS restriction enzyme table.

For a limited time, try a sample of BsaI-HFv2 and see how it works for you!

1. Potapov, V. et. al. “Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.” ACS Synth. Biol. 2018. DOI: 10.1021/acssynbio.8b00333.

2. Pryor, J.M. et al (2020) “Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design.” PloS One, https://doi.org/10.1371/journal.pone.0238592

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 [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications.

Featured Products:

	NEB^®Golden Gate Assembly Kit (BsmBI-v2) Contains an optimized mix of BsmBI-v2 (optimized for Golden Gate Assembly) and T4 DNA Ligase. Can direct the accurate assembly of 2 – 20+ inserts/modules using the Golden Gate approach.
	Esp3I An isoschizomer of BsmBI that is recommended for use at 37°C and is supplied with CutSmart Buffer, adding flexibility to your reaction setup
	BsaI-HFv2 New version of BsaI-HF that is optimized for Golden Gate Assembly.
	BsmBI-v2 New version of BsmBI that is optimized for Golden Gate Assembly.
	PaqCI New Type IIS restriction enzyme with 7 basepair recognition sequence (AarI isoschizomer).

Featured Protocols:

Videos

Golden Gate Assembly Workflow

Find out how Golden Gate Assembly can be used to quickly join multiple DNA fragments.
Restriction Enzymes in Golden Gate Assembly

Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for re-assembly. View a list of TypeIIS enzymes.
Golden Gate Assembly Domestication Tutorial

This video discusses Domestication, or the removal of Type IIS cut sites naturally occurring in vector or insert sequences, as it relates to Golden Gate Assembly.
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.