Rapid Screening of Recombinant Plasmids

Abstract

Construction of recombinant plasmid DNA is one of the cornerstones of molecular biology. The ability to clone DNA in a plasmid vector opens doors to downstream applications such as amplification of DNA, expression of desired genes, and construction of DNA libraries. Recombinant plasmids are generally constructed by first isolating the target DNA and linearizing the plasmid vector of choice (see Chapter 2). The insert DNA is subsequently ligated to the vector DNA (see Chapters 15 and 17) and the ligation mixture transformed into an appropriate Escherichia coli host (see Chapters 3–5) (1,2). E. coli transformed with recombinant plasmid or self-ligated vector will both grow on appropriate selective medium; therefore, screening is almost always necessary to select colonies containing the recombinant plasmid and, in some cases, with the correct orientation of insert.