TABLE 2.

Strategy used for verification of rocF mutants by PCR and sequence analyses

Strain	PCR primer pairsa		PCR-sequencing
	Up, bpb	Down, bp	Up	Down
N6 WT	R13+K4, none	R5+K8, none; R17+K8, none	NAc	NA
			NA	NA
N6 rocF::aphA3	R13+K4, 680	R5+K8, 860	NDd	R5+K8e
N6 236-2	R13+K4, 890	R17+K8, 250	R13+H16	R17+K8
SS1 WT	R13+K4, none	R17+K5, none; R17+K8, none	NA	NA
			NA	NA
SS1 rocF::aphA3	R13+K4, 680	R17+K5, 1160	ND	R5+K8
SS1 236-2	R13+K4, 890	R17+K8, 250	R13+H16	R17+K8

^aPrimer pairs used for PCR are listed. Primers are schematically depicted in Fig. 1. The first primer of the pair is specific for rocF, whereas the second primer is specific for the kanamycin resistance gene. The primer sequences are given in Table 1. The results for the PCR reactions from columns 2 and 3 of the table are shown in Fig. 2B. 

^bUp and down, rocF regions immediately up- or downstream, respectively, of insertion sites for kanamycin cassettes. The sizes of the PCR products (shown in Fig. 2B) are given in base pairs. 

^cNA, not applicable. 

^dND, not determined. 

^eUnderlining indicates primers used for direct sequencing of PCR products.