DescriptionTth Endonuclease IV is a thermostable apurinic/apyrimidinic (AP) endonuclease from Thermus thermophilus. Tth Endo IV will hydrolyze an AP site at the first phosphodiester bond 5' to the lesion leaving a 3' hydroxyl and a deoxyribose 5'-phosphate at the 5' terminus. The enzyme also has a 3'-diesterase activity.
Product SourceAn E. coli strain that carries the cloned Thermus thermophilus endonuclease IV gene.
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|ThermoPol® Reaction Buffer||-20||10X|
Advantages and Features
- Alkaline elution (1)
- Alkaline unwinding (2)
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme required to cleave 10 pmol of a 60-mer oligonucleotide duplex containing a single AP site* in a total reaction volume of 10 μl in 1 hour at 65°C.
* An AP site is created by treating 10 pmol of a 60-mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.
1X ThermoPol® Reaction Buffer
Incubate at 65°C
1X ThermoPol® Reaction Buffer:
20 mM Tris-HCl
10 mM (NH4)2SO4
10 mM KCl
2 mM MgSO4
0.1% Triton® X-100
pH 8.8 @ 25°C
10 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
0.1% Triton® X-100
pH 7.4 @ 25°C
Unit Assay Conditions1X ThermoPol Reaction Buffer containing 5 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl.
Quality Control AssaysThe 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.
- Enzyme stability above 80°C is assured by adding ZnCl2 to a final concentration of 25 μM in the reaction.
- Diluent Compatibility: Diluent D
- Pflaum, M. et al. (1998). Free Rad. Res.. 29, 585-594.
- Hartwig, A. et al. (1996). Toxicology Letters. 88, 85-90.