Product Class: Kit

NEB® Golden Gate Assembly Kit (BsaI-HF®v2)
Item# Description List Price Web Price Qty
E1601L NEB Golden Gate Assembly Kit (BsaI-HFv2) - 100 reactions $600.00
$540.00
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E1601S NEB Golden Gate Assembly Kit (BsaI-HFv2) - 20 reactions $225.00
$202.50
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*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 (20+ fragments)

    Product Introduction

    • 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

    Product Information

    Description

    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.

    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.

    pGGA is a 2,714 bp cloning vector useful for Golden Gate Assembly. The plasmid contains two BsaI sites; digestion with BsaI releases a 41 bp fragment and a 2,133 bp vector backbone fragment to receive your insert or assembly.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    T4 DNA Ligase Reaction Buffer -20 10X
    NEB Golden Gate Assembly Mix
    pGGA Destination Plasmid -20
    Product Categories:
    DNA Assembly, Cloning and Mutagenesis Kits Products
    Applications:
    DNA Assembly and Cloning,
    Golden Gate Assembly

    Advantages and Features

    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

    Storage Temperature

    -20°C

    References

    1. Engler, C. et al (2008). PLoS ONE. 3: e3647.
    2. Engler, C. et al (2009). PLoS ONE. 4: e5553.
    3. Lee, J.H. et al (1996). Genetic Analysis: Biomolecular Engineering. 13; 139-145.
    4. Padgett, K.A. and Sorge, J.A. (1996). Gene. 168, 31-35.
    5. Weber, E. et al (2001). PLoS ONE. 6; e19722.
    6. Christian, M. et al (2010). Genetics. 186, 757-761.
    7. Potapov, V. et al. (2018). bioRxiv. 322297.

    Protocols, Manuals & Usage

    Protocols

    1. Golden Gate Assembly Protocol for Using NEB Golden Gate Assembly Kit (BsaI-HFv2) (E1601)
    2. Transformation Protocol for Using NEB Golden Gate Assembly Kit (BsaI-HFv2) (E1601)
    3. Recommended Screening Protocols for Using NEB Golden Gate Assembly Kit (BsaI-HFv2)

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

    Usage & Guidelines

    • Insert Considerations When Using NEB Golden Gate Assembly Kit (BsaI-HFv2) (NEB #E1601)
    • Technical Tips For Optimizing Golden Gate Assembly Reactions

    Application Notes

    Tools & Resources

    Selection Charts

    • Synthetic Biology/DNA Assembly Selection Chart

    Web Tools

    Faqs & Troubleshooting

    FAQs

    1. Why does the Golden Gate Assembly Mix now feature BsaI-HFv2?
    2. How does NEB Golden Gate Assembly work?
    3. What affects the efficiency of Golden Gate Assembly?
    4. Why do many of the published Golden Gate Assembly articles feature precloned inserts as opposed to inserts generated by PCR?
    5. Using amplicons directly without precloning seems much easier, but is theassembly efficiency decreased?
    6. Can PCR amplicons be used directly in assembly reactions without purification?
    7. For amplicon inserts, why are the calculations suggested as using the overallpGGA destination plasmid length and the overall amplicon lengths? Shouldn’tonly the part of pGGA functioning as the vector backbone be used?
    8. Why is Golden Gate also used for single insert cloning?
    9. What if there is an internal BsaI site(s) in my inserts?
    10. Why is there a 60°C, 5 min heat step at the end of the assembly reaction?
    11. How can I minimize PCR-generated errors in my amplicon inserts?
    12. I’m doing a moderate (4–5 insert) assembly but don’t have access to a thermal cycler. Can I use the simpler protocol using the 1 hr 37°C incubation?
    13. Can the Golden Gate Assembly reactions be scaled down?
    14. Can I use other competent E. coli strains than NEB 10-beta? Can I use subcloning efficiency cells?
    15. I would like to use colony PCR to screen my transformants and sequence myassembly. Are there any recommended sequencing or colony PCR primers?

    Troubleshooting

    Tech Tips

    • Use of the NEB Golden Gate Assembly Tool (GoldenGate.neb.com) is strongly recommended; this tool will check insert sequences for internal BsaI sites and design primers to amplify your inserts for Golden Gate Assembly. The primers will feature 6 bases at the 5´ end flanking the BsaI recognition site, the recognition site itself, plus the 4-base unique 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 improved accuracy (7). This ligase fidelity information can be used in conjunction with the NEB Golden Gate Assembly Kit (BsaI-HFv2) to achieve high efficiency and accurate complex assemblies. Please visit www.neb.com/GoldenGate for more information and examples of 24 fragment assemblies with >90% accuracy

    • Two basic protocol approaches exist for assembly—constant 37°C single temperature incubations, or cycling protocols alternating between 37°C (optimal temperature for endonuclease digestion within a temperature range for ligase stability), and 16°C (optimal temperature for ligation). The assembly protocol suggestions are based on the number of inserts in your assembly reaction, but there is considerable flexibility; match the protocol to your desired efficiency of assembly and scheduling needs. In general: a. Assembly of 1-4 inserts does not require cycling; single 37°C incubations work well. b. Cycling assemblies using 1 min temperature steps work well for multiple inserts. c. Cycling assemblies using 5-10 min temperature steps work well for larger scale assemblies (>10 inserts) and for any assembly for which maximal assembly yields and transformation levels are desired. d. Regardless of the number of inserts, if it is more convenient, any Golden Gate Assembly can be done overnight with 30 cycles of 37°C, 5 min → 16°C, 10 min; i.e., there is no downside for longer protocols being used.

    • For precloning of inserts, we recommend using the NEB PCR Cloning Kit as the kit's pMiniT 2.0 vector backbone has no BsaI sites present.
    • The standard protocol using 30 cycles (alternating between 37°C and 16°C) results in high levels of accurate assemblies with low background, even for 24 fragment assemblies. BsaI-HFv2 and T4 DNA Ligase however are very stable and they will continue to function up to 60 cycles. 

    • While BsaI and BsaI-HFv2 are blocked by overlapping dcm methylation (methylation at the C5 position of cytosine in the sequences CCAGG or CCTGG), this is usually not an issue for Golden Gate Assembly. Commonly used destination vectors are designed to avoid upstream CC(A or T) bases in front of the BsaI GGTCTC recognition site that would create an overlapping dcm methylation site.
     

    Quality & Safety

    Quality Assurance Statement

    Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

    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 DataSheets

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Legal and Disclaimers

    This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

    While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

    For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

    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.