New England Biolabs, M0305S, Endonuclease V

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 deoxyinosine site* in a total reaction volume of 10 μl in 1 hour at 37°C. * A deoxyinosine site is synthetically prepared with a dl in the middle of one strand of a 34 mer oligonucleotide duplex. Reaction Conditions 1X NEBuffer™ 4 Incubate at 37°C 1X NEBuffer™ 4 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 1 mM DTT (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 250 mM NaCl 0.1 mM EDTA 50% Glycerol 0.15% Triton® X-100 1 mM DTT 200 µg/ml BSA pH 8 @ 25°C Heat Inactivation 65°C for 20 minutes Unit Assay Conditions 1X NEBuffer 4 containing 10 pmol of fluorescently labeled oligonucleotide duplex in a total volume of 10 μl. FAQ Q: What is the molecular weight of Endonuclease V? A: The molecular weight of the NEB Endonuclease V is 80 kDa because it is produced as an MBP fusion (MBP MW = 55kDa) Q: What substrate is used to test Endonuclease V activity? A: The susbstrates we use to test enzyme activity are listed under the unit definition of each enzyme. In the case of Endonuclease V a 34-mer oligonucleotide duplex containing a single deoxyinosine site is used. Q: What is the activity of Endonuclease V in the different NEBuffers? A: The activity of Endonuclease V in the NEBuffers is as follows: NEBuffer 1: 75% activity; NEBuffer 2: 50% activity; NEBuffer 3: 100% activity; NEBuffer 4: 100% activity. Q: How well does Endonuclease V cleave at U:A sites? A: Endonuclease V does not work well at U:A sites. However, it will cleave the phosphodiester bond 3' to a deoxyuridine mismatch if it is a U:G mismatch. For additional information please refer to the "DNA Repair Glycosylases on Various Damages Bases" page in our website. Q: Can Endonuclease V cleave the loop in a stem-loop DNA structure? A: Yes, Endonuclease V has been reported to be able to cleave the loop in a stem-loop DNA structure. Q: Can a nick generated by Endonuclease V activity be repaired with DNA ligase? A: Yes, a nick generated by Endonuclease V can be ligated by DNA ligase. Q: Can Endonuclease V recognize and nick mismatches? A: Yes. Endonuclease V will recognize mismatches. However, the efficiency of the nicking in these sites is very low. Q: What is the difference between Endonuclease V (#M0305) and Thermostable Endonuclease Q (#M0701)? A: Although both Endonuclease V and Thermostable Endonuclease Q recognize deoxyinosine in DNA substrates that result in the formation of a nick with a 3′-OH and a 5′-phosphate, the location of the nick is different. Endonuclease V cleaves the second phosphodiester bond on the 3′ side of the deoxyinosine; Thermostable Endonuclease Q, cleaves on the phosphodiester bond immediately 5′ to the deoxyinosine. In addition, Endonuclease V activity is optimal at 37°C and can be heat-inactivated; Thermostable Endonuclease Q activity is optimal between 65°C and 85°C and cannot be heat-inactivated.