New England Biolabs, M0304S, Endonuclease IV

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. *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 NEBuffer™ 3 Incubate at 37°C 1X NEBuffer™ 3 100 mM NaCl 50 mM Tris-HCl 10 mM MgCl2 1 mM DTT (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 250 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol 0.15% Triton® X-100 pH 7.4 @ 25°C Heat Inactivation 85°C for 20 minutes Unit Assay Conditions 1X NEBuffer 3 containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total reaction volume of 10 μl. FAQ Q: What is the activity of Endonuclease IV in the NEBuffers? A: The Endo IV optimum buffer is 1X NEBuffer 3. It has no activity in NEBuffer 1, 100% in NEBuffer 2 and 25% activity in NEBuffer 4. Q: What is the molecular weight for Endonuclease IV? A: The molecular weight of Endonuclease IV is 31kDa. Q: Does Endonuclease IV cleave ssDNA? A: Yes, Endonuclease IV cleaves abasic sites on both ssDNA and dsDNA. Q: Will the 5' terminus left by an Endonuclease IV cleavage be suitable for subsequent ligation by T4 DNA ligase? A: The 5' end generated by Endonuclease IV cleavage is not suitable for ligation. Q: In addition to AP sites, does Endonuclease IV have cleavage activity on other types of DNA damage? A: Endo IV also recognizes 5,6-dihydrothymine. However, its activity on this substrate is less than on AP sites. For more information please refer to this page in our website: (DNA Repair Enzymes). Q: Can an abasic site be created and then cleaved with Endonuclease IV in the same reaction? How active is Uracil-DNA-Glycosylase (cat# M0280S) in Endonuclease IV buffer? A: The Endonuclease IV buffer is NEBuffer 3. UDG is 100% active in NEBuffer 3 and therefore it is possible to perform both reactions in NEBuffer3.