FIELD: biochemistry.
SUBSTANCE: reaction is started by mixing solutions of the DNA probe and cell extract in a 1:1 ratio and the change in the fluorescence intensity of the FRET pair is recorded in the time interval of 15–3,600 sec. Solutions of substrate (20 µM), buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 1 mM DTT, 7% glycerol) and sample of cell extract of the microorganism (20 μg of total protein) are mixed in a 96-well fluorometer plate. A complementary oligodeoxyribonucleotide carrying the FAM/BHQ1 FRET pair at the ends of the chain and also containing a 6-mer degenerate sequence is used as a DNA probe.
EFFECT: simplifying the method and increasing the productivity of determining the activity of restriction endonucleases in cell extracts of microorganisms.
4 cl, 3 dwg, 2 ex
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