Complement this kit with NEBridge Golden Gate Assembly Kit (BsaI-HFv2) (NEB #E1601)
The NEBridge Golden Gate Assembly Kit (BsmBI-v2) contains an optimized mix of BsmBI-v2 and T4 DNA Ligase. BsmBI-v2 has been engineered by NEB and outperforms BsmBI in Golden Gate Assemblies. Together these enzymes can direct the assembly of multiple inserts/modules and also single insert/library generation cloning with single insert(s) using the Golden Gate approach. The pGGAselect destination plasmid is also provided, which provides a backbone for your assembly. This versatile destination construct has flanking recognition sites in the correct orientation for BsmBI-directed assemblies, and also BsaI- and BbsI-directed assemblies, enabling the destination plasmid to conveniently be used with all three of the most commonly used Type IIS restriction enzymes used for Golden Gate Assembly. It features convenient restriction enzyme sites for subcloning, and has T7/SP6 promoter sequences to enable in vitro transcription.
The efficient and seamless assembly of DNA fragments, commonly referred to as Golden Gate Assembly (1,2), had its origins in 1996, when for the first time it was shown that multiple inserts could be assembled into a vector backbone using only the sequential (3) or simultaneous (4) activities of a single Type IIS restriction enzyme and T4 DNA Ligase.
Type IIS restriction enzymes bind to their recognition sites but cut the DNA downstream from that site at a positional, not sequence- specific, cut site. Thus, a single Type IIS restriction enzyme can be used to generate DNA fragments with unique overhangs. As an example, BsmBI has a recognition site of CGTCTC(N1/N5), where the CGTCTC represents the recognition/binding site, and the N1/N5 indicates the cut site is one base downstream on the top strand, and five bases downstream on the bottom strand. Assembly of digested fragments proceeds through annealing of complementary four base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites, so no further cutting is possible. The assembly product accumulates with time.
While particularly useful for multi-fragment assemblies such as Transcription Activator Like Effectors (TALEs)(5) and TALEs fused to a FokI nuclease catalytic domain (TALENs)(6), the Golden Gate method can also be used for cloning of single inserts and inserts from diverse populations that enable library creation, and multi-site mutagenesis involved in directed evolution (7).
Please note that while general descriptions regarding Golden Gate Assembly use the BsmBI nomenclature, this kit and protocols feature the specific engineered form optimized for Golden Gate Assembly, BsmBI-v2.
To learn more about the Golden Gate Assembly workflow, watch this video tutorial.
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