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  • T4 PDG (T4 Endonuclease V)

    Description

    T4 PDG (pyrimidine dimer glycosylase) has both DNA glycosylase and APlyase activity. The 16 kd protein recognizes cis-syn-cyclobutane pyrimidine dimers caused by UV irradiation. The enzyme cleaves the glycosyl bond of the 5´ end of the pyrimidine dimer and the endonucleolytic activity cleaves the phosphodiester bond at the AP site.

    Highlights

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

    Product Source

    Purified from an E. coli strain carrying a plasmid encoding T4 denV gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T4 PDG Reaction Buffer10X
    BSA-2010 mg/ml

    Advantages and Features

    Applications

    • DNA damage studies
    • Single cell gel electrophoresis (comet assay)

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that catalyzes the conversion of 0.5 µg of UV irradiated supercoiled pUC19 DNA to > 95% nicked plasmid in a total reaction volume of 20 µl in 30 minutes at 37°C. Nicking is assessed by agarose gel electrophoresis. Irradiated plasmid contains an average of 3-5 pyrimidine dimers.

    Reaction Conditions

    1X T4 PDG Reaction Buffer
    Supplement with 100X BSA
    Incubate at 37°C

    1X T4 PDG Reaction Buffer:
    100 mM NaCl
    1 mM DTT
    1 mM EDTA
    25 mM Na2HPO4
    pH 7.2 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    100 mM NaCl
    0.1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.5 @ 25°C

    Heat Inactivation

    No

    Unit Assay Conditions

    1X T4 PDG Reaction Buffer containing 0.5 µg of UV irradiated supercoiled pUC19 DNA, supplemented with 100 µg/ml BSA in a 20 µl reaction.

    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.
    • 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. For best results incubation time should be 30 minutes or less.
    2. Addition of 1 µl of phenol to the sample before loading will improve electrophoresis by stripping the protein from the DNA.
    3. Warm buffer to room temperature as it precipitates at 4°C.

    References

    1. Higgins, K.L. and Lloyd, R.S. (1987). Mutation Research. 183, 117-121.

    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 T4 PDG in the NEBuffers 1-4?
    2. What is the molecular weight of T4 PDG?
    3. Is T4 PDG a tagged protein?
    4. Can the T4 PDG be used in the comet assay?
    5. Does T4 PDG remove the damaged base?
    6. What type of damaged DNA does T4 PDG recognize?

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