Subcloning Strategies and Protocols

Subcloning procedures are used to transfer DNA fragments from one vector context (plasmid, cosmid, or phage) to another. They are commonly used to construct expression systems, and to transfer fragments into specialized vectors for the preparation of hybridization probes and single-stranded DNA sequencing templates. Typically, cloning vectors have three essential elements, an antibiotic resistance marker, an origin of replication (to allow selection of transformed E. coli host cells), and one or more restriction sites into which foreign DNA may be inserted. Phage vectors, such as those based on M13 (1 ), do not use antibiotic markers; instead transformants produce virus particles that kill or slow the growth of infected cells, and appear as plaques on lawns of uninfected bacteria. We describe here a variety of general subcloning strategies and protocols; discussions of more specialized strategies may be found elsewhere (e.g., ref. 2 ).