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  • Tma Endonuclease III

    Description

    A thermostable homolog of the E. coli Endonuclease III (Nth) (1). It acts as an N-glycosylase and an AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating an apurinic (AP site). The AP-lysase activity then cleaves the resulting abasic site. 

    Tma Endonuclease III recognizes abasic sites, 5,6 dihydroxythymine and thymine glycol in DNA (2).


    Product Source

    Cloned from Thermotoga maritima and overexpressed in E. coli.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ThermoPol® Reaction Buffer-2010X

    Advantages and Features

    Applications

    • Alkaline elution (3)
    • Alkaline unwinding (4)

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave 1 pmol of a 60-mer oligonucleotide duplex containing a single AP site* in a total reaction volume of 10 µl in 1 hour at 65°C. 

    *An AP site is created by treating 10 pmol of a 60-mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C. .

    Reaction Conditions

    1X ThermoPol® Reaction Buffer
    Incubate at 65°C

    1X ThermoPol® Reaction Buffer:
    20 mM Tris-HCl
    10 mM (NH4)2SO4
    10 mM KCl
    2 mM MgSO4
    0.1% Triton® X-100
    pH 8.8 @ 25°C

    Usage Concentration

    10,000 units/ml

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    100 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1% Triton® X-100
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    Unit Assay Conditions

    1X ThermoPol Reaction Buffer containing 5 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 µl.

    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, DNA):
      The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA as determined by agarose gel electrophoresis.

    Notes

    1. Diluent Compatibility: Diluent D
      100 mM KCl, 10 mM Tris-HCl (pH 7.4 @ 25°C), 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100 and 50% glycerol.

    References

    1. Nelson, K.E. et al. (1999). Nature. 399
    2. Marks, K. unpublished observation.
    3. Pflaum, M. et al. (1998). Free Rad. Res. 29
    4. Hartwig, A. et al. (1996). Toxicol. Lett. 88

    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. What is the activity of Tma Endonuclease III in the NEBuffers 1-4?
    2. What is the activity of Tma Endonuclease III in ThermoPol buffer?
    3. What is the molecular weight of Tma Endonuclease III?
    4. Is Tma Endonuclease III a tagged protein?
    5. What substrate is used to test the Tma Endonuclease III activity?
    6. What abasic sites does Tma Endonuclease III recognize?

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