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A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome
C Barth1, D J Fraser, P R Fisher
1School of Microbiology, La Trobe University, Bundoora 3083, Melbourne, Australia.
Abstract:
A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed. It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells. This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP). The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant.