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The Uracil Cleavage System provides two enzymes, which, when added sequentially to a reaction containing a synthetic DNA fragment containing a deoxy-uracil, generate a single nucleotide gap at the location of the uracil residue. The system consists of two individual enzyme components, Uracil DNA Glycosylase (UDG) and Endonuclease VIII, provided at a 10X concentration to be added to a reaction containing a uracil-containing polynucleotide sequence. UDG catalyses the excision of the uracil base, creating an abasic site with an intact phosphodiester backbone (1,2). The lyase activity of Endonuclease VIII breaks the phosphodiester backbone both 3′ and 5′ to the abasic site, liberating the deoxyribose sugar (3,4).
Source of ProteinEach component protein is purified separately from E. colistrains containing recombinant Endonuclease VIII and Uracil-DNA Glycosylase genes.
Supplied with:UDG (G5010)10 mM Tris-HCl50 mM NaCl1 mM DTT0.1 mM EDTA50% glycerolpH 7.5 @ 25°C
Endonuclease VIII (Y9080):10 mM Tris-HCl250 mM NaCl0.1 mM EDTA50% glycerolpH 8.0 @ 25°C
Unit Definition, UDG1 unit is defined as the amount of enzyme that catalyzes the release of 1.8 nmol of uracil in 30 minutes from double-stranded, tritiated, uracil containing-DNA at 37°C in 1X UDG Reaction Buffer.
Unit Definition, Endonuclease VlllOne unit is defined as the amount of enzyme required to cleave 1 pmol of an oligonucleotide duplex containing a single AP site in 1 hour at 37°C.
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