New England Biolabs, M0299L, Endonuclease VIII

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 VIII 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 VIII Reaction Buffer Incubate at 37°C 1X Endonuclease VIII Reaction Buffer 10 mM Tris-HCl 75 mM NaCl 1 mM EDTA (pH 8 @ 25°C) Storage Buffer 10 mM Tris-HCl 250 mM NaCl 0.1 mM EDTA 50% Glycerol pH 8 @ 25°C Heat Inactivation 75°C for 10 minutes Unit Assay Conditions 1X Endonclease VIII Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl. FAQ Q: What is the activity of Endonuclease VIII in NEBUffers? A: Endo VIII has 75% activity in NEBuffers 1 and 4, and 50% activity in NEBuffer 2. The use of NEBuffer 3 is not recommended. Q: Does Endonuclease VIII cleave RNA? A: Endonuclease VIII does not show any activity on RNA. Q: Does treatment of DNA with Endonuclease VIII leave a 5' phosphate? A: Yes. After Endonuclease VIII digestion, DNA can be directly ligated without having to treat with a kinase. Q: What damaged bases does Endonuclease VIII recognize? A: The damaged bases recognized by Endonuclease VIII are: urea, 5, 6- dihydroxythymine, thymine glycol, 5-hydroxy-5- methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea. Q: Will Endonuclease VIII work in rCutSmart buffer? A: Yes, Endonuclease VIII 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.