Note: Orientation A and B refer to the conventions used in Ellermeier et al (2002, in press, Gene). Each set of primers is designed to precisely replace the open reading frame with the template insertion (starting at P1/P4 and ending at P4/P1). These primers are all designed with 40-nt homology regions, and 20 nt annealing. The screening primers (5'pcr and 3'pcr) are homologous to the 20 nt directly flanking the 40-nt homology regions on each end of the deletion. They have not been tested for quality in any way.

Note: I've posted a protocol for doing this technique in Salmonella on our protocols page.