The NEBridge Golden Gate Assembly Kit (BsaI-HFv2) contains an optimized mix of BsaI-HFv2 andT4 DNA Ligase. Together these enzymes can direct the assembly of multiple inserts/modules using theGolden Gate approach. Also included is the pGGAselectdestination plasmid, which provides a backbonefor your assembly, features convenient restriction enzyme sites forsubcloning, and has T7/SP6 promoter sequences to enable in vitro transcription.The efficient and seamless assembly of DNA fragments, commonly referred to asGolden Gate assembly (1,2), has its origins in 1996, when for the first time it wasshown that multiple inserts could be assembled into a vector backbone using onlythe sequential (3) or simultaneous (4) activities of a single Type IISrestriction enzymeand T4 DNA Ligase.Type IIS restriction enzymes bind to their recognition sites but cut the DNAdownstream 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 withunique overhangs. As an example, BsaI has a recognition site of GGTCTC(N1/N5), where the GGTCTC represents the recognition/binding site, and the N1/N5 indicates the cut site is one base downstream on the top strand, and fivebases downstream on the bottom strand. Assembly of digested fragmentsproceeds through annealing of complementary four base overhangs on adjacentfragments. The digested fragments and the final assembly no longer containTypeIIS 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 TranscriptionActivator Like Effectors (TALEs)(5) and TALEs fused to a FokI nuclease catalyticdomain (TALENs)(6), the Golden Gate method can also be used for cloning ofsingle inserts and inserts from diverse populations that enable library creation. To learn more about the Golden Gate Assembly workflow, watch this video tutorial. To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.

Please note that while general descriptions regarding Golden Gate Assembly use the BsaI nomenclature, this kit and protocols feature the specific engineered form optimized for Golden Gate Assembly, BsaI-HFv2.

Figure 1: Overview: Assembly Protocol of Golden Gate Assembly

Figure 2: Golden Gate Workflow

Figure 3: Golden Gate Assembly of 24 fragments can be achieved with high efficiency and accuracy

Figure 4.

This product is related to the following categories:

DNA Assembly, Cloning and Mutagenesis Kits Products

This product can be used in the following applications:

DNA Assembly and Cloning,

High-throughput cloning and automation solutions,

NEBridge® Golden Gate Assembly

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