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  • Taq DNA Ligase

    Description

    Taq DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini of two adjacent oligonucleotides which are hybridized to a complementary target DNA. The ligation will occur only if the oligonucleotides are perfectly paired to the complementary target DNA and have no gaps between them; therefore, a single-base substitution can be detected. Taq DNA Ligase is active at elevated temperatures (45°C-65°C) (1,2).

    Highlights

    • Isolated from a recombinant source
    • Thermostable ligase for incorporation of phosphorylated oligonucleotides during PCR and Ligase Chain Reaction
    • Taq DNA Ligase is NOT a substitute for T4 DNA Ligase
    • Supplied with 10X Reaction Buffer containing NAD+

    Product Source

    Purified from an E. coli strain containing the cloned ligase gene from Thermus aquaticus HB8 (1).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Taq DNA Ligase Reaction Buffer-20*10X
    λ DNA-BstEII Digest-20500 μg/ml

    * Reagents' Storage Notes

    • Taq DNA Ligase Reaction Buffer: For long term storage (>30 days), store at -20°C.

    Advantages and Features

    Applications

    • Allele-specific gene detection using Ligase Detection Reaction and Ligase Chain Reaction (1,3).
    • Mutagenesis by incorporation of a phosphorylated oligonucleotide during primer extension amplification (4).

    Properties and Usage

    Unit Definition

    (Cohesive End Unit)
    One unit is defined as the amount of enzyme required to give 50% ligation of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA in a total reaction volume of 50 µl in 15 minutes at 45°C.

    Reaction Conditions

    1X Taq DNA Ligase Reaction Buffer
    Incubate at 45°C

    1X Taq DNA Ligase Reaction Buffer:
    20 mM Tris-HCl
    25 mM Potassium Acetate
    10 mM Magnesium Acetate
    1 mM NAD 1
    10 mM DTT
    0.1% Triton® X-100
    pH 7.6 @ 25°C

    Usage Concentration

    40,000 units/ml

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    Unit Assay Conditions

    1X Taq DNA Ligase Reaction Buffer and DNA (20 µg/ml). After incubation at 45°C for 15 minutes, the reaction is terminated by addition of stop dye (50% glycerol, 50 mM EDTA and bromophenol blue), heated at 70°C for 10 minutes and then loaded on a 0.7% agarose gel. Due to the presence of ligase, the cos ends of BstEII-digested λ DNA will stay together after 70°C heat treatment.

    Quality Control

    Quality Assurance Statement

    • Each lot is tested for contaminating single-stranded DNA exonuclease, endonuclease, ribonuclease and phosphatase activities.

    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.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

    Notes

    1. Reaction Conditions: Incubate DNA and enzyme in 1X Taq DNA Ligase Buffer at 45°C for 15 minutes or in a thermocycler with a program suited to the reaction described by Barany (1991) Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase. Proc. Natl. Acad. Sci. USA 88, 189-193. The reaction is stopped with a mixture of 50% glycerol, 50 mM EDTA, bromphenol blue."
    2. 1X Taq DNA Ligase Reaction Buffer requires NAD+ as a cofactor. NAD+ is supplied in the 10X Taq DNA Ligase Reaction Buffer; the buffer should be stored at -70°C to extend the half life of the NAD+ cofactor.

    References

    1. Barany, F. (1991). Proc. Natl. Acad. Sci. USA. 88, 189-193.
    2. Takahashi, M. et al. (1984). J. Biol. Chem.. 259, 10041-10047.
    3. Barany, F. (1991). The Ligase Chain Reaction in a PCR World. 5-16.
    4. Michael, S.F. (1994). Biotechniques. 16, 411-412.

    Supporting Documents

    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]

    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. Is Taq DNA Ligase used for a special technique?
    2. How much ligation occurs at mismatches when using Taq DNA Ligase?
    3. How many temperature cycles will Taq DNA Ligase survive?
    4. Can Taq DNA Ligase be used for cloning?
    5. What is the molecular weight of Taq DNA Ligase?
    6. What is the concentration of Taq DNA Ligase?
    7. Why is the Taq DNA Ligase buffer brown?
    8. Does Taq DNA Ligase require NAD?
    9. What is the activity of Taq DNA Ligase in other NEBuffers?
    10. What is the activity of Taq DNA Ligase at various temperatures?
    11. What is the stability of Taq DNA Ligase at 95°C?
    12. What is the stability of Taq DNA Ligase at room temperature?
    1. Protocol for Taq DNA Ligase (M0208)

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