Determination of plasmid copy number in yeast transformants by means of agarose plugs

The determination of plasmid copy number in Saccharomyces cerevisiaetransformants containing circular or linear plasmids is currently performed with total yeast DNA extracts obtained from cultures grown under selection. The determination is based essentially on quantitative Southern hybridization of an appropirate probe to a sequence present both on plasmid and chromosomal DNA in digested or undigested samples run out on conventional agarose gels. The DNA extraction procedure calls for treatment of cell lysates with organic solvents that could entail systemic losses of eithr plasmid or chromosomal DNA thus producing artifactual results. We propose here a method based on the assumption that quantitative analysis of plasmid and chromosomal DNA extracted from yeast cells embedded in agarose plugs will furnish more reliable results. With this procedure the cells are lysed in situ, thus avoiding possible losses of material, and the chromosomes and plasmid DNAs, trapped within the agarose matrix, can be separated by pulse field electrophoresis.