New England Biolabs, M0268S, Endonuclease III (Nth)

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to cleave 1 pmol of a 34 mer oligonucleotide duplex containing a single AP site* in a total reaction volume of 10 μl in 1 hour at 37°C in 1X Endonuclease III Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex. * An AP site is created by treating 10 pmol of a 34 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 Endonuclease III (Nth) Reaction Buffer Incubate at 37°C 1X Endonuclease III (Nth) Reaction Buffer 20 mM Tris-HCl 1 mM EDTA 1 mM DTT (pH 8 @ 25°C) Usage Concentration 10,000 units/ml Storage Buffer 10 mM Tris-HCl 250 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 Heat Inactivation 65°C for 20 minutes Unit Assay Conditions 1X Endonuclease III (Nth) Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplexes in a total reaction volume of 10 μl FAQ Q: What is the activity of Endonuclease III in the NEBuffer 1-4? A: Endonuclease III is fully active in all four buffers. Q: What is the molecular weight of Endonuclease III? A: The molecular weight of Endonuclease III is 23 kDa. Q: What types of damaged bases are recognized and removed by Endonuclease III? A: Some of the damaged bases recognized and removed by Endo III are urea, 5, 6 dihydroxythymine, thymine glycol, 5-hydroxy-5 methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihdrothimine and methyltartronylurea. Q: Is Endonuclease III a tagged protein? A: No. Endo III is not a tagged protein. Q: What substrate is used to test Endonuclease III activity? A: The substrates used to test enzyme activity are described in the unit definition of each enzyme. For Endonuclease III the substrate used is a 34-mer oligonucleotide duplex containing a single AP site. Q: Will Endonuclease III work in rCutSmart buffer? A: Yes, Endonuclease III will work in rCutSmart buffer. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart.