PCR primers used for screening opa genes following transformation of Neisseria meningitidis.

Following each transformation, three PCR reactions were carried out at each opa locus to confirm recombination. The antibiotic resistance cassette was amplified using internal primers to confirm it had inserted into the genome. The antibiotic resistance cassette was amplified with one internal primer and one primer within a locus-specific gene adjacent to opa to confirm insertion had occurred within the target opa gene. Finally, the entire opa locus was amplified using primers in adjacent genes to confirm a double crossover event had occurred.*wt = wild-type. Primer sequences were: kan-if, 5′-AGCCATATTCAACGGGAAAC-3′; kan-ir, 5′-TTTGCTTTGCCACGGAAC-3′; tetF, 5′-TTGATGCTCTTGATCTTCC-3′; tetR, 5′-TAACAGCAAACAGTAATGG-3′; NMB1464-7SalI, 5′-TGCAGAGTCGACGGCATCAACACCCATGC-3′; NMB1466-0SacII, 5′-CCGCCTCCGCGGTTATGTTGTGCGACCAGTCC-3′; kan-5-out, 5′-TCAAAAATATGGTATTGATAATCC-3′; kan-3-out, 5′-TGTAACATCATTGGCAACGC-3′. Other primer sequences are described in table 1.