NEB® Golden Gate Assembly Kit (BsaI-HF®v2)
- Updated to include BsaI-HFv2 (optimized for Golden Gate)
- Seamless cloning – no scar remains following assembly
- Includes destination plasmid with T7/SP6 promoters
- Ordered assembly of multiple fragments (2-20+) in a single reaction
- Can also be used for cloning of single inserts and library preparations
- Efficient with regions with high GC content and areas of repeats
- Compatible with a broad range of fragment sizes (< 100 bp to > 15 kb)
- Free tool available at GoldenGate.neb.com
- Free Ligase Fidelity Viewer to determine mismatch ligation with 4-bp overhangs
Golden Gate Assembly Workflow
The NEB Golden Gate Assembly Kit (BsaI-HFv2) contains an optimized mix of BsaI-HFv2 and T4 DNA Ligase. Together these enzymes can direct the assembly of multiple inserts/modules using the Golden Gate approach. Also included is the pGGA destination plasmid, which provides a backbone for your assembly, 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), has 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, 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 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. 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.
The following reagents are supplied with this product:
Store at (°C) Concentration T4 DNA Ligase Reaction Buffer -20 10 X NEB Golden Gate Enzyme Mix (BsaI-HFv2) -20 pGGA Destination Plasmid -20
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