Isolation of restriction fragments from large plasmids recovered from bacteria with multiple plasmids

A rapid and simple method for isolation of restriction DNA fragments from large plasmids is described. The loss of large plasmids is avoided by restriction endonuclease cleavage in an agarose gel before DNA precipitation. Plasmids were separated in low-melting-point agarose by electrophoresis, the desired plasmid DNA band was cut from the gel and digested with a restriction endonuclease in the agarose. Restriction fragments in agarose were recovered by a modified phenol-extraction, concentrated with 2-butanol and precipitated with ethanol. The procedure simplifies the task of cloning genes from large plasmids, resulting in high yields of restriction fragments from a desired plasmid in a short time.