cultivate the organism of interest to large numbers (e.g., 1010 bacterial cells) from which plasmid DNA chromosomal DNA, or total cellular DNA may be isolated and purified for endonudease digestion.
Following digestion, fragments are subjected to agarose gel electrophoresis, which allows them to be separated according to their size differences as previously described for Southern hybridization (see Figure 8-4, B). During electrophoresis all nudeic add fragments of the same size comigrate as a single band. For many digestions, electrophoresis results in the separation of several different fragment sizes (see Figure 8-15). The nucleic acid bands in the agarose gel are stained with the fluorescent dye ethidium bromide, which allows them to be visualized on exposure to UV light. Stained gels are photographed for a permanent record (Figures 8-16 to 8-18).
One variation of this method, known as ribo-typing, involves enzymatic digestion of chromosomal DNA followed by Southern hybridization using probes for genes that encode ribosomal RNA. Because all bacteria contain ribosomal genes, a hybridization pattern will be obtained with almost any isolate, but the pattern will vary depending on the arrangement of genes in a particular strain's genome.
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