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  • Uracil-DNA Glycosylase (UDG)

    Description

    E. coli Uracil-DNA Glycosylase (UDG) catalyses the release of free uracil from uracil-containing DNA. UDG efficiently hydrolyzes uracil from single-stranded or double-stranded DNA, but not from oligomers (6 or fewer bases).


    Highlights

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

    Product Source

    An E. coli strain that carries the UDG gene from E. coli.

    Advantages and Features

    Applications

    • Treatment of 0.1 µg of uracil-containing DNA with 1 unit of UDG for 10 minutes at 37°C renders the DNA incapable of being copied by DNA polymerase. The enzyme can be 95% heat killed by incubation at 95°C for 10 minutes. Since UDG remains partially active following heat treatment at 95°C, it is recommended that uracil glycosylase inhibitor be added to prevent degradation of product DNA. Alternatively, reaction products can be immediately extracted with phenol/chloroform.

    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 37°C. 

    Reaction Conditions

    1X UDG Reaction Buffer
    Incubate at 37°C

    1X UDG Reaction Buffer:
    20 mM Tris-HCl
    1 mM DTT
    1 mM EDTA
    pH 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

    Unit Assay Conditions

    1X UDG Reaction Buffer, 1 unit of uracil DNA Glycosylase, 0.2 µg 3H-uracil DNA (104 -105 cpm/µg) for 30 minutes at 37°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. UDG is active over a broad pH range with an optimum at pH 8.0, does not require divalent cation, and is inhibited by high ionic strength (> 200 mM).

    References

    1. Lindahl, T. et al. (1977). J. Biol. Chem.. 252, 3286-3294.
    2. Wang, Z. et al. (1991). Gene. 99, 31-37.
    3. Devchand, P.R. et al. (1993). Nucl. Acids Res.. 21, 3437-3443.

    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 UDG in the NEBuffers 1-4?
    2. Will UDG work in T4 DNA ligase buffer?
    3. What is the molecular weight of UDG?
    4. Is UDG a tagged protein?
    5. I see that UDG works optimally at 37°C. Do you have another glycosylase that works at higher temperatures?
    6. Does the Uracil Glycosylase inhibitor (UGI) inhibit UDG?
    7. Does UDG cut RNA?
    8. Can UDG be used to remove dU from a short 21mer oligo? Do the dU residues need to be spaced in any special way to avoid problems with cleavage?
    9. Are there any specific recommendations for the use of UDG on single stranded DNA? The materials on the web and data card seem to describe conditions for use with dsDNA.
    10. What is the difference between UDG and UNG?
    11. Does UDG release uracil from ss and dsDNA?

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