Rapid preparation and identification of insert-containing recombinant plasmid DNA.

The identification of transformant bacterial colonies containing potential recombinant DNA clones can be performed using a wide variety of screening methods including colony hybridization and autoradiography, polymerase chain reaction (PCR) and restriction endonuclease analysis of individual plasmid DNA (1,5). While colony hybridization can efficiently screen large numbers of recombinant clones, many investigators avoid using radioisotopes for both safety and financial considerations. In addition, PCR may not be cost-effective for the individual screening of large numbers of transformants, including recombinant clones generated by differential display or for library amplification. Several minipreparation methods for the isolation of plasmid DNA have been described using the standard or modified versions of the alkaline lysis procedure (3,4,6–9) or the boil-extraction procedure (2). Plasmid DNA that can be used for downstream applications such as restriction endonuclease digestion or automated DNA sequenc-