Preparation and FIGE separation of infrequent restriction fragments fromMycoplosnw mycoidesDNA

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Abstract
Use of procedures for obtaining satisfactory preparation and digestion of intact DNA of Mycoplasma mycoides subsp. mycoides Y in agarose blocks is reported. The use of inverted field agarose gel electrophoresis (FIGE) for separation of the small number of fragments derived from the genome by several restriction endonuclease digestions is shown. An effect that fragments containing replication forks remain in the well during FIGE, distorting the representative yield of restriction fragments on the gels, is overcome by incubating cells with chloramphenicol for 1 1/2 h before harvest to allow rounds of replication to go to completion without new initiations of DNA synthesis.