The NEBridge 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 pGGAselect 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.
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 In its simplest form, Golden Gate Assembly requires a Type IIS recognition site, in this case, BsaI-HFv2 (GGTCTC), added to both ends of a dsDNA fragment. After digestion, these sites are left behind, with each fragment bearing the designed 4-base overhangs that direct the assembly.
Figure 3: Golden Gate Assembly of 24 fragments can be achieved with high efficiency and accuracy Twenty-four fragment assemblies of the lacI/lacZ cassette were performed using the protocol included in this article. While 30 cycles is sufficient to achieve 24 fragment assemblies, the stability of the BsaI-HFv2 and T4 DNA Ligase allows continued assembly through 45 and 60 cycles with a low background. (a) Efficiency of assembly and (b) accuracy of assembly versus cycle number.
Figure 4.
pGGA is a 2,200 bp cloning vector useful for Golden Gate Assembly. The plasmid contains two BsaI sites; digestion with BsaI releases a 87 bp fragment and a 2,133 bp vector backbone fragment to receive your insert or assembly.
This product is related to the following categories:
Lee, J.H. et al (1996). Genetic Analysis: Biomolecular Engineering. 13; 139-145.
Padgett, K.A. and Sorge, J.A. (1996). Gene. 168, 31-35.
Weber, E. et al (2001). PLoS ONE. 6; e19722.
Christian, M. et al (2010). Genetics. 186, 757-761.
Potapov, V. et al. (2018). Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synth. Biol. 7, 11, 2665-2674. DOI: 10.1021/acssynbio.8b00333,
Use of the NEB Golden Gate Assembly Tool (GoldenGate.neb.com) is strongly recommended; this tool will check insert sequences for internal Type IIS restriction enzyme sites and design primers to amplify your inserts for Golden Gate Assembly. The primers will feature 6 bases at the 5′ end flanking the recognition site, the recognition site itself, plus the 4-base overhangs that determine correct annealing and ligation of the inserts. All overhangs will automatically be designed as non-palindromic (to eliminate self-insert ligations), unique, and in the correct orientations to ensure correct assembly.
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 will result in greater accuracy (Potapov, V. et al. (2018) ACS Synth. Biol., 7, 2665–2674.). This ligase fidelity information can be used in conjunction with the NEB Golden Gate Assembly kits to achieve high efficiency and accurate complex assemblies. Please visit www.neb.com/GoldenGate for more information.
NEB has developed ligase fidelity tools to facilitate the design of high-fidelity Golden Gate Assemblies:
Standard Golden Gate protocol suggests using 30 cycles, alternating between restriction and cutting. BsaI-HFv2, BsmBI-v2 and T4 DNA Ligase are very stable, allowing cycling up to 60 cycles, with high efficiency and fidelity. Consider whether your workflow would be enhanced by adding more cycles.
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