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  • Blunt/TA Ligase Master Mix

    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.

    * 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.

    Advantages and Features

    Applications

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

    Properties and Usage

    Storage Temperature

    -20°C

    Heat Inactivation

    No

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Functional Test (Ligation and Transformation):
      The product is tested for ligation and transformation efficiency using DNA containing blunt or cohesive termini. Ligation products are transformed and must exceed specifications for efficiency (transformants/µg).

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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 info@neb.com or fill out the Technical Support Form for appropriate document.
    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?
    1. Ligation Protocol for Cloning with Blunt/TA Ligase Master Mix (M0367)
    2. Transformation Protocol (M0367)

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