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DNA Modifying Enzymes and Cloning >
Nucleases >
Tth Endonuclease IV |
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Description:
Tth 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.
Source:
An E. coli strain that carries the cloned Thermus thermophilus endonuclease IV gene.
Applications:- Alkaline elution (1)
- Alkaline unwinding (2)
Reagents Supplied:
ThermoPol Reaction Buffer
Enzyme Properties
Nuclease Properties Comparison
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
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
Unit Definition:
One unit is defined as the amount of enzyme required to cleave 1 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.
Unit Assay Conditions: 1X ThermoPol Reaction Buffer containing 5 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl.
Concentration:
10,000 units/ml
Storage Conditions:
10 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Diluent Compatibility: Vent Diluent
Usage notes:- Enzyme stability above 80°C is assured by adding ZnCl2 to a final concentration of 25 μM in the reaction.
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 Tth Endonuclease IV 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 Tth Endonuclease IV with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Exonuclease Activity::
Incubation of a 50 μl reaction containing 100 units of Tth Endonuclease IV in NEBuffer 1 with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/μg) for 4 hours at 37°C released < 0.1% of the total radioactivity.
References
- Pflaum, M. et al. (1998) Free Rad. Res., 29, 585-594.
- Hartwig, A. et al. (1996) Toxicology Letters, 88, 85-90.
Reagents Sold Separately
ThermoPol Reaction Buffer
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