A large-scale preparative polyacrylamide gel electrophoresis (PAGE) system for the isolation of high-purity supercoiled plasmid-DNA is described. This method should prove suitable for the isolation of large DNA molecules, either plasmid or linear DNA, that is required for the production of transgenic animals, for instance. The efficiency of the method is illustrated by the isolation of the gene for the green fluorescent protein, cloned into a mammalian expression vector and used for transfection of eukaryotic cells.