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    Item# Description List Price Web Price Qty
    M0367L Blunt/TA Ligase Master Mix - 250 rxns $557.00
    $501.30
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    M0367S Blunt/TA Ligase Master Mix - 50 rxns $139.00
    $125.10
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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
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    Product Introduction

    Blunt/TA Ligase Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer. This master mix is specifically formulated to improve ligation and transformation of both blunt-end and single-base overhang substrates.

    • Simplifies reaction set-up, ensures an optimized ratio of enzyme and buffer components
    • No thawing is necessary as it remains liquid during storage at -20°C
    • Ligations for subcloning can be carried out in small volumes with low DNA concentrations, allowing users to conserve precious DNA samples and directly transform many strains of chemically competent E. coli without dilution
    • Not sure which ligase to choose? Refer to our DNA and RNA Ligase Properties Chart

    Product Information

    Description

    Blunt/TA Ligase Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer. This master mix is specifically formulated to improve ligation and transformation of both blunt-end and single-base overhang substrates. The master mix format simplifies reaction set-up, ensures an optimized ratio of enzyme and buffer components, and yields robust, rapid ligation of all types of DNA ends using a short incubation time at room temperature. No thawing is necessary as it remains liquid during storage at -20°C.* Ligations for subcloning can be carried out in small volumes with low DNA concentrations, allowing users to conserve precious DNA samples and directly transform many strains of chemically competent E. coli without dilution.

    Learn more about Joining Difficult to Ligase dsDNA Fragments and Efficient Adaptor Ligation for the Preparation of dsDNA Libraries using Blunt/TA Ligase Master Mix.

    Blunt/TA Ligase Master Mix improves yields for ends that typically react slowly


    Yields of final ligation product for all reaction conditions using high concentration T4 DNA Ligase (NEB #M0202), the Quick Ligation Kit (NEB #M2200) and Blunt/TA Master Mix (NEB #M0367). Nick, cohesive end and 3´ single-base overhang substrates were incubated for 15 minutes; the 5´ single-base overhang was incubated for 1 hour.
    * Freezers vary in their actual internal temperature. Our testing demonstrates that the master mix is liquid at -20°C. Freeze-thaw testing at -70°C has confirmed that the performance is unchanged after 20 freeze/thaw cycles.

    M0367
    Ligation reactions with single-base overhang vector and insert were set-up using the Blunt/TA Ligase Master Mix and incubated for different times at 25°C (A) or at different temperatures for 15 minutes (B). Two microliters of each reaction were used to transform a 50 μl aliquot of NEB 10-beta Competent E. coli (NEB #C3019). Transformants resulting from triplicate plating 50 μl of a 1:5 dilution of the outgrowth were counted and graphed. The results indicate that the Blunt/TA Ligase Master Mix works well at 25°C, and is complete in 15 minutes.

    Product Source

    Purified from an E. coli strain containing a recombinant gene encoding T4 DNA Ligase.

    Reaction Volume Definition

    1X Blunt/TA Ligase Master Mix with DNA substrates in a 10 μl reaction volume incubated at 25°C. A 10 μl reaction contains 1,800 cohesive end units of T4 DNA Ligase.
    Product Categories:
    DNA Ligases Products
    Applications:
    Cloning Ligation

    Advantages and Features

    Application Features

    • Vector construction
    • Linker ligation
    • Fragment assembly
    • Library construction
    • TA cloning

    Properties and Usage

      Storage Temperature

      -20°C

      Heat Inactivation

      No

      Notes

      1. DNA: Purified DNA for ligations can be dissolved in dH2O (Milli-Q® water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the amount of vector DNA should be 20–100 ng and the insert should be added at a 3-fold molar excess. For ligation volumes greater than 10 μl, increase the volume of Blunt/TA Ligase Master Mix such that it remains 50% of the reaction. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.
      2. Time and Temperature: Most ligations performed using the Blunt/TA Ligase Master Mix reach an end point at 60 minutes or less when performed between 4–37°C. Incubation beyond this time provides no additional benefit. Our recommendation for a 25°C (room temperature) incubation was chosen after evaluation of performance at 4°C, 16°C, 25°C, and 37°C. Most conditions reached at least 50% performance within 15 minutes. Shorter times can also be used.
      3. Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates with the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes.
      4. Electroporation: While electroporation can dramatically increase transformation efficiency, Blunt/TA Ligase Master Mix is not directly compatible with transformation by electroporation. It is necessary to reduce the PEG concentration. We recommend purification of the ligated DNA by spin column. 
      5. Biology: Some DNA sequences are not easy to clone. Sequences that form structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.

      References

      1. Quick J., Quinlan, A.R., Loman N.J. (2014). A reference bacterial genome dataset generated on the MinION™ portable single-molecule nanopore sequencer. Gigascience. 3, 22. PubMedID: 25386338,
      2. Madoui M.A., Engelen S., Cruaud C., Belser C., Bertrand L., Alberti A., Lemainque A., Wincker P., Aury J.M. (2015). Genome assembly using Nanopore-guided long and error-free DNA reads. BMC Genomics. 16, 327. PubMedID: 25927464,
      3. Karamitros T., Magiorkinis G. (2015). A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits. Nucleic Acids Res.. 15, 43. PubMedID: 26240383,
      4. Karlsson E., Lärkeryd A., Sjödin A., Forsman M., Stenberg P. (2015). Scaffolding of a bacterial genome using MinION nanopore sequencing. Sci Rep.. 5, 11996. PubMedID: 26149338,

      Faqs & Tech Tips

      FAQs

      1. My transformations using Blunt/TA Ligase Master Mix reactions produced no colonies. What happened?
      2. Can the Blunt/TA Ligase Master Mix be used for the ligation of sticky-end fragments?
      3. Can the ligation reaction produced by the Blunt/TA Ligase Master Mix be directly used to transform electrocompetent cells?
      4. The recommended volume for a Blunt/TA Ligase Master Mix reaction is 10ul. I like to set-up my ligation reactions in a 20 ul volume. Can I scale-up the reaction?
      5. I routinely use more than 5 ul of my ligation reactions to transform 50 ul aliquots of competent cells. When I do this with the Blunt/TA Ligase Master Mix my transformation plates have very few colonies. What do you think is the problem?
      6. Can I incubate a Blunt/TA Ligase Master Mix ligation reaction for longer than 15 minutes?
      7. Can I incubate a Blunt/TA Ligase Master Mix ligation reaction at a temperature other than 25°C?
      8. My Blunt/TA Ligase Master Mix has frozen in my freezer. Is this a problem?
      9. The protocol for Blunt/TA Ligase Master Mix indicates the reactionshould not be heat inactivated. How can I inactivate the ligationactivity?

      Protocols & Manuals

      Protocols

      1. Ligation Protocol for Cloning with Blunt/TA Ligase Master Mix (M0367)
      2. Transformation Protocol (M0367)

      Datacards

      The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)

      Other Tools & Resources

      Selection Charts

      • DNA Ligase Selection Chart
      • Properties of DNA and RNA Ligases
      • Substrate-based Ligase Selection Chart

      Usage & Guidelines

      • Tips for Maximizing Ligation Efficiencies

      Troubleshooting Guides

      Troubleshooting Tips for Ligation Reactions

      Interactive Tools

      Application Notes

      Citations

      • Yamagishi, J., et al. (2017) Serotyping dengue virus with isothermal amplification and a portable sequencer Scientific Reports; 7(1), e3510. PubMedID: 28580104

      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.

      Blunt/TA Ligase Master Mix