With constant advances both in the development of new enzymes (e.g., BsaI-HFv2) 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 20+ 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). Their research has enabled complex fragment assemblies with high efficiencies and >90% accuracy. Use Ligase Fidelity Viewer to visualize overhang ligation preferences for T4 DNA ligase 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 introduce two new restriction enzymes for use in Golden Gate: Esp3I, an isoschizomer of BsmBI that is recommended for use at 37°C and is supplied with CutSmart® Buffer, and the improved BsaI-HFv2 and BsmBI-v2, which are optimized for Golden Gate Assembly.
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. . DOI: 10.1021/acssynbio.8b00333.
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|NEB® Golden Gate Assembly Kit (BsaI-HF®v2)
Contains an optimized mix of BsaI-HFv2 (optimized for Golden Gate Assembly) and T4 DNA Ligase. Can direct the accurate assembly of 2 – 20+ inserts/modules using the Golden Gate approach.
An isoschizomer of BsmBI that is recommended for use at 37°C and is supplied with CutSmart Buffer, adding flexibility to your reaction setup
New version of BsaI-HF that is optimized for Golden Gate Assembly.
Find out how Golden Gate Assembly can be used to quickly join multiple DNA fragments.
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.
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.