Following transformation of a ligation reaction into competent E. coli cells, successful subclones are conventionally identified by two methods. The first involves preparing “mini-prep” plasmid DNA from a number of colonies and then identifying the desired recombinant plasmid on the basis of either its unique restriction enzyme digest pattern or by direct DNA sequencing. The second, often used when large numbers of putative recombinants are involved, is by colony hybridization with a labeled probe. In this chapter an alternative PCR-based method for direct screening of transformants is described that is both facile and rapid, completely circumventing time-consuming DNA plasmid preparations. In its simplest form described below, transformed colonies are directly tooth-picked into a small volume of PCR reaction mix that includes primers that flank the cloning site. This is usually achieved with Universal primers from the vector polylinker (1 ). Following in vitro amplification, aliquots of each reaction are analyzed by agarose gel electrophoresis, which reveals both the presence and size of cloned inserts.