Product Class: Nuclease

T4 PDG (T4 Endonuclease V)

recombinant unique buffer incubation temp heat inactivation no bsa
Catalog #SizeConcentration
M0308S2,000 units10,000 units/ml
M0308L10,000 units10,000 units/ml

Description

Highlights

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

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.

Heat Inactivated

No

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.

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.

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.
  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?