Endonuclease VIII (C007B-555776)

Aladdin's Endonuclease VIII is a recombinant protein expressed in E. coli and purified on the PerfectProtein™ Platform developed by aladdin. It is a DNA damage repair enzyme with both DNA N-glycosylase and AP-lyase activities. The N-glycosylase activity cuts and releases damaged pyrimidines on dsDNA to produce a Apurinic (AP) site, and the AP lyase activity cleaves DNA phosphodiester backbone at the 3' and 5' ends of the AP site, excising the AP site and creating a nucleotide DNA gap with 3' and 5' phosphate termini.Impaired bases that can be recognized and excised by Endonuclease VIIIApplication：DNA damage and repair studies; single-cell gel electrophoresis; alkaline elution; DNA alkaline unwinding; identification and excision of damaged pyrimidines from dsDNA; cleavage of 3' and 5' of the AP site to create a nucleotide DNA gap with 3' and 5' phosphate termini.Definition of enzyme activity unit: One unit is defined as the amount of enzyme required to cleave 1pmol of a 34 mer oligonucleotide duplex containing a single AP site in a total reaction volume of 10μl in 1 hour at 37ºC in 1X Endonuclease VIII Reaction Buffer containing 10pmol of fluorescently labeled oligonucleotide duplex.Purity: ≥ 95%. This product is free of RNase, other DNA exonucleases and endonucleases.Enzyme storage buffer: 10mM Tris-HCl (pH8 at 25ºC), 250mM NaCl, 0.1mM EDTA, 1mM DTT, 50% (v/v) Glycerol.Inactivation or inhibition: This product can be inactivated by incubation at 75ºC for 10 minutes.10X Endonuclease VIII Buffer：100mM Tris-HCl, 750mM NaCl, 10mM EDTA, pH 8 at 25ºC.Enzyme storage buffer：10mM Tris-HCl (pH8 at 25ºC), 250mM NaCl, 0.1mM EDTA, 1mM DTT, 50% (v/v) glycerol.Inactivation or inhibition：This product can be inactivated by incubation at 75ºC for 10 minutes.10X Endonuclease VIII Buffer：100mM Tris-HCl, 750mM NaCl, 10mM EDTA, pH 8 at 25ºC.Precautions：Keep this product on ice when handling and store at -20ºC immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Excision of AP site in dsDNAa. Preparation of dsDNA: Anneal ssDNA (containing one or more dU bases in the middle) and its complementary ssDNA (without dU bases in the middle) with 's Annealing Buffer for DNA Oligos (5X) according to manufacture’s instructions.b. Preparation of dsDNA with AP site. Treat annealed dsDNA products with ’s Uracil-DNA Glycosylase (E.coli) /Uracil-DNA Glycosylase (Heat-labile, Bacterium) /Uracil-DNA Glycosylase (Heat-labile, Cod) to generate dsDNA with AP site.c. Excision of AP sites in dsDNA. (a) Set up the reaction on ice as follows:Component Volume Nuclease-free Water 15µl 10X Endonuclease VIII Buffer 2µl dsDNA with AP site (10µM/µl) 2µl Endonuclease VIII (10U/µl) 1µl Note: Endonuclease VIII should be added at last after mixing well all other components. After adding Endonuclease VIII, mix the reaction well by pipetting. To digest large amounts of DNA, extend the digestion time or scale up the reaction volume proportionally.(b) Incubate at 37ºC for 30 min.(c) Terminate the reaction by heating at 75ºC for 10 minutes or incubating on ice for 1-2 minutes followed by the addition of 2X RNA Loading Buffer (, R0215).2. For other applications, please refer to relevant literature.

Specification: ≥95%, This product is free of RNase, other DNA exonucleases and endonucleases.