Verification of the ΔpqsC mutant by PCR and Southern. A) Verification by PCR. To confirm that the pqsC gene is mutated, two primer pairs Fupout-pqsC/Rin-kan and Fupout-pqsC/Rdownout-pqsC were used, with Fupout-pqsC and Rdownout-pqsC hybridizing outside the DNA region used for the recombination event. Primers Fupout/Rin-kan allow DNA amplification only if the kanamycin cassette is inserted, while the primer pair Fupout/Rdownout amplify a fragment of different size when mutant or wild type genomic DNA is used. B) Verification by Southern. The HindIII-digested total genomic DNA of three ΔpqsC generated mutants was hybridized using the kanamycin cassette as a probe. One band of 8016 bp is obtained when the cassette is inserted into pqsC. The HindIII-digested pUC4K and PA14 genomic DNA was used as positive and negative controls, respectively.
Lesic and Rahme BMC Molecular Biology 2008 9:20 doi:10.1186/1471-2199-9-20