Product Class: Nuclease

Tth Endonuclease IV

recombinant unique buffer incubation temp heat inactivation no
Catalog #SizeConcentration
M0294S500 units10,000 units/ml
M0294L2,500 units10,000 units/ml

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.


Product Source

An E. coli strain that carries the cloned Thermus thermophilus endonuclease IV gene.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
ThermoPol® Reaction Buffer-2010X

Advantages and Features

Applications

  • Alkaline elution (1)
  • Alkaline unwinding (2)

Properties and Usage

Unit Definition

One 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.

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

Usage Concentration

10,000 units/ml

Storage Temperature

-20°C

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

Heat Inactivation

No

Unit Assay Conditions

1X ThermoPol Reaction Buffer containing 5 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl.

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.
  • 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.

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. Enzyme stability above 80°C is assured by adding ZnCl2 to a final concentration of 25 μM in the reaction.
  2. Diluent Compatibility: Diluent D

References

  1. Pflaum, M. et al. (1998). Free Rad. Res.. 29, 585-594.
  2. Hartwig, A. et al. (1996). Toxicology Letters. 88, 85-90.
  1. How does Tth Endonuclease IV perform in PCR?