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  • 9°N™ DNA Ligase

    Description

    9°N™ 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. 9°N DNA Ligase is active at elevated temperatures (45°C-90°C).

    Highlights

    • Can be used to ligate nicks in DNA while incubating at high temperatures
    • Extremely thermostable and can withstand PCR conditions
    • Will not ligate short 4 base overlaps (typical of restriction enzyme digests), while it efficiently ligates 12 base pair overlaps
    • Isolated from a recombinant source

    Product Source

    Purified from an E. coli strain containing the cloned ligase gene from the extremely thermophilic marine archaea Thermococcus sp.(strain 9°N). The archaea was isolated from a submarine thermal vent, at a depth of 2,500 meters, 9° north of the equator at the East Pacific Rise (1).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    9 N DNA Ligase Reaction Buffer (10X)10X

    Advantages and Features

    Applications

    • Allele-specific gene detection using Ligase Detection Reaction and Ligase Chain Reaction (2,4).
    • Mutagenesis by incorporation of a phosphorylated oligonucleotide during PCR amplification (5).

    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.

    A cohesive end unit is equivalent to the nick-closing unit (1).

    Reaction Conditions:
    1X 9°N DNA Ligase Reaction Buffer

    1X 9°N DNA Ligase Reaction Buffer:
    10 mM Tris-HCl
    600 µM ATP
    2.5 mM MgCl2
    2.5 mM Dithiothreitol
    0.1 % Triton X-100
    pH 7.5 @ 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
    10 mM ammonium sulfate

    Heat Inactivation

    No

    Unit Assay Conditions

    1X 9°N DNA Ligase Reaction Buffer and 20 µg/ml BstEII-digested λ DNA in a 50 µl reaction. 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 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. 9°N DNA Ligase is not a substitute for T4 DNA Ligase. The cohesive end unit is equivalent to the nick-closing unit of Barany et al.
    2. Incubate DNA and enzyme in 1X 9°N DNA Ligase Reaction 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.

    References

    1. Thermococcus sp. (strain 9°N-7) isolated by Dr. Holger Jannasch (1991). Woods Hole Oceanographic Institute.
    2. Barany, F. (1991). Proc. Natl. Acad. Sci. USA. 88, 189-193.
    3. Takahashi, M. et al. (1984). J. Biol. Chem.. 259, 10041-10047.
    4. Barany, F. (1991). The Ligase Chain Reaction in a PCR World. (pp. 5-16, Cold Spring Harbor: Cold Spring Harbor Laboratory.
    5. Michael, S.F. (1994). Biotechniques. 16, 411-412.

    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. Protocol for 9°N DNA Ligase (M0238)

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