M.CfrBI activity was tested by incubation of MTase with bacteriophage φ80 vir DNA. A typical assay was carried out in a 50 μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 10 mM EDTA, 5 mM 2-mercaptoethanol, 80 μM S-adenosyl-L-methionine, 1 μg λ DNA and 2.5 units of His6-M.CfrBI. The reaction mixture was incubated at 37 °C for 1 h. After methylation, DNA was extracted with phenol/chloroform and precipitated with ethanol. The pellet was re-dissolved in a 20-μl reaction buffer and incubated for 1 h at 37 °C with CfrBI. The resulting digested products were analysed by 1% agarose gel electrophoresis; methylation was tested by the absence of digestion.
A quantitative analysis of M.CfrBI activity was based on the incorporation of tritiated methyl groups into substrate DNA. The methylation reactions were carried out as above, except that the reaction mixtures contained labelled 10 μCi [3H] SAM (85 Ci/mM, Amersham) and a PvuII DNA fragment instead of φ80 vir DNA. After incubation, the DNA fragment was digested with corresponding ENase. The digested products were loaded onto 2.5% low-melting agarose gel, excised and purified. Then these samples were adsorbed onto 2,5 mm Whatman DE81 paper circles, dipped in 95% ethanol, dried and counted in a liquid scintillator cocktail [18] (link).
A quantitative analysis of M.CfrBI activity was based on the incorporation of tritiated methyl groups into substrate DNA. The methylation reactions were carried out as above, except that the reaction mixtures contained labelled 10 μCi [3H] SAM (85 Ci/mM, Amersham) and a PvuII DNA fragment instead of φ80 vir DNA. After incubation, the DNA fragment was digested with corresponding ENase. The digested products were loaded onto 2.5% low-melting agarose gel, excised and purified. Then these samples were adsorbed onto 2,5 mm Whatman DE81 paper circles, dipped in 95% ethanol, dried and counted in a liquid scintillator cocktail [18] (link).