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
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

Learn about Ligase Fidelity and Push the Limits of Golden Gate Assembly (50+ fragments)

  • 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 (2-50+) 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
  • Try our Ligase FidelityTools (for the design of high-fidelity Golden Gate Assemblies)
Featured Videos
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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.

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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  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
  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


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,
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.
Quality Control Assay
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Specifications
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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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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