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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
T4 PDG (T4 Endonuclease V) |
 | | | | T4 PDG (T4 Endonuclease V) | | | |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
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.
Source:
Purified from an E. coli strain carrying a plasmid encoding T4 denV gene.
Applications:- DNA damage studies
- Single cell gel eletrophoresis (comet assay)
Reagents Supplied:
T4 PDG Reaction Buffer (10X)
BSA (100X)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X T4 PDG Reaction Buffer
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X T4 PDG Reaction Buffer:
100 mM NaCl
1 mM DTT
1 mM EDTA
25 mM Na2PO4
pH 7.2 @ 25°C
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.
Concentration:
10,000 units/ml
Storage Conditions:
10 mM Tris-HCl
0.1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- For best results incubation time should be 30 minutes or less.
Addition of 1 µl of phenol to the sample before loading will improve electrophoresis by stripping the protein from the DNA.
Warm buffer to room temperature as it precipitates at 4°C.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA (HindIII digest)
and 100 units of T4 PDG (T4 Endonuclease V) incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of T4 PDG (T4 Endonuclease V) with 1 μg of
nonirradiated pUC19 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
References
- Higgins, K.L. and Lloyd, R.S. (1987) Mutation Research, 183, p. 117-121.
Reagents Sold Separately
BSA
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