NEBridge® Golden Gate Assembly Kit (BsaI-HF® v2)

Catalog #	Concentration	Size	List Price	Quantity	Your Price
E1601L		100 reactions	$661.00		$594.90
E1601S		20 reactions	$248.00		$223.20

Catalog #	Size	List Price	Your Price
E1601L	100 reactions	$661.00	$594.90
E1601S	20 reactions	$248.00	$223.20

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.

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.

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.

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.

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.

Reagents Supplied

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

E1601S -20

NEBridge^® Golden Gate Enzyme Mix (BsaI-HFv2)	M2616AVIAL	-20	1 x 0.02 ml
T4 DNA Ligase Reaction Buffer	B0202SVIAL	-20	1 x 1 ml	10 X
pGGAselect DNA	N0309AAVIAL	-20	1 x 0.1 ml	75 ng/µl

E1601L -20

NEBridge^® Golden Gate Enzyme Mix (BsaI-HFv2)	M2616AAVIAL	-20	1 x 0.1 ml
T4 DNA Ligase Reaction Buffer	B0202SVIAL	-20	1 x 1 ml	10 X
pGGAselect DNA	N0309AAVIAL	-20	1 x 0.1 ml	75 ng/µl

Features

Seamless cloning – no scar remains following assembly
Ordered assembly of multiple fragments in a single reaction
Can also be used for cloning of single inserts
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
2 year shelf life

Properties & Usage

Materials Required but not Supplied

User-defined inserts
Competent cells
Other materials for transformation

Companion Products

References

Engler, C. et al (2008). PLoS ONE. 3: e3647.
Engler, C. et al (2009). PLoS ONE. 4: e5553.
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,

Protocols

Usage Guidelines

DataCards

manualE1601

Selection Charts

Synthetic Biology/DNA Assembly Selection Chart

Web Tools

FAQs

Tech Tips

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.
vNEB has developed ligase fidelity tools to facilitate the design of high-fidelity Golden Gate Assemblies:
- Ligase Fidelity Viewer-visualize overhang ligation preferences
- GetSet™ – predict high-fidelity junction sets
- SplitSet™ – split DNA sequence for scarless high-fidelity assembly
All tools are available at neb.com/research/NEBeta-tools
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.

Citations

Quality Control Assay

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Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety Data Sheets

Legal And Disclaimer

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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