Eco72I (PmaCI)

E. coli that carries the cloned eco72IR gene from E. coli RFL72\n\nDiluent Buffer:\n10mM Tris-HCl (pH 7.4 at 25 degrees C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.\n\nStorage Buffer:\n10mM Tris-HCl (pH 7.4 at 25 degrees C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol.\n\nUnit Definition:\nOne unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37 degrees C in 50ul of assay buffer.\n\nOverdigestion Assay: \nNo detectable change in the specific fragmentation pattern is observed after 10-fold overdigestion (10u/ug lambda DNA x 1 hour) with Eco72I (see Star Activity).\n\nLigation/Recutting Assay: \nAfter 10-fold overdigestion (5units/ug DNA x 2 hours) with Eco72I, more than 80% of the DNA fragments can be ligated in a reaction mixture containing 20-40u of T4 DNA ligase/1ug of fragments and 10% PEG at a 5'-termini concentration of 0.03uM. More than 90% of these can be recut.\n\nStar Activity:\nAn excess of enzyme (15u/ug DNA x 1 hour) or low salt concentration may result in star activity. \n\nLabeled Oligonucleotide (LO) Assay: No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of restriction endonuclease for 4 hours.\n \nBlue/White Cloning Assay: The mix of pUC57/HindIII, pUC57/Eco32I and pUC57/PstI digests was incubated with 10 units of enzyme for 16 hours. After religation and transformation 0.1% of white colonies were detected.\n\nMethylation Effects: Eco72I does not cut CAm5CGTG. Blocked by CG methylation.\n\nStability during Prolonged Incubation: A minimum of 0.5 units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37 degrees C. \n\nThermal Inactivation: Enzyme is inactivated by incubation at 65 degrees C for 20min.\n\nDigestion of Agarose-embedded DNA: A minimum of 5 units of enzyme is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.\n\nNumber of Recognition Sites in DNA: Lambda: 3, PhiX174: 0, M13mp18/19: 0, pBR322: 0, pUC18/19: 0, pUC57: 0,\npTZ19R/U: 0\n\nStorage:\nStore at -20 degrees C

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