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  • Afu Uracil-DNA Glycosylase (UDG)

    Description

    A thermostable homolog of the E. coli Uracil-DNA Glycosylase (UDG) (1,2) from Archaeoglobus fulgidus. Afu UDG catalyzes the release of free uracil from uracil-containing DNA. Afu UDG efficiently hydrolyzes uracil from single-stranded or double-stranded DNA.

    Highlights

    • Isolated from a recombinant source 
    • Supplied with 10X Reaction Buffer

    Product Source

    Cloned and overexpressed in E. coli.

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracil-containing DNA. Activity is measured by release of [3H]-uracil in a 50 µl reaction containing 0.2 µg DNA (104-105 cpm/µg) in 30 minutes at 65°C.

    Reaction Conditions

    1X ThermoPol® II (Mg-Free) Reaction Buffer
    Incubate at 65°C

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

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    No

    Molecular Weight

    Theoretical: 22717 daltons

    Specific Activity

    96 units/mg

    Unit Assay Conditions

    1X ThermoPol II (Mg-free) Reaction Buffer, 1 unit of Afu Uracil-DNA Glycosylase, 0.2 µg 3H-uracil DNA (104-105 cpm/µg) for 30 minutes at 65°C in a total reaction volume of 50 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):
      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.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Notes

    1. Afu UDG retains 50% activity in the presence of 150 mM NaCl. Afu UDG retains less than 1% activity after boiling for 30 minutes in standard reaction conditions.
    2. Under standard reaction conditions, uracil glycosylase inhibitor (UGI) does not inhibit Afu UDG.

    References

    1. Sandigursky, M. and Franklin, W. A. (1999). Current Biology. 9
    2. Sandigursky, M. and Franklin, W. A. (2000). J. Biol. Chem.. 275

    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. What is the activity of Afu UDG in the NEBuffer 1-4?
    2. Will Afu UDG work in Fpg buffer?
    3. Will Afu UDG work in T4 DNA ligase or Thermopol Buffer?
    4. What is the molecular weight of Afu UDG?
    5. Is Afu UDG a tagged protein?
    6. I see that Afu UDG works optimally at 65°C. Do you have another glycosylase that works at lower temperatures?
    7. Does the Uracil Glycosylase inhibitor (UGI) inhibit Afu UDG?
    8. Does Afu UDG release uracil from ss and dsDNA?
    9. Does Afu UDG cut RNA?
    10. Are there any specific recommendations for the use of UDG on single stranded DNA?

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