Verification of Gene Deletions

<div><p>(A) Verification of <i>ddrA</i> and <i>recA</i> gene deletions by PCR analysis. Purified PCR fragments were amplified from the genomic DNA of strains R1, TNK104, TNK106, and TNK110 using primers that flank the coding sequences for <i>ddrA</i> and <i>recA.</i> Products were separated on a 0.8% agarose gel to establish whether the fragment size corresponded to the gene-replacement cassette. The left panel depicts the replacement of <i>ddrA</i> in TNK104 and TNK110. The right panel depicts the replacement of <i>recA</i> in TNK106 and TNK110. Expected sizes of the wild-type and mutant sequences are given in the figure above each image of the agarose gel.</p> <p>(B) Verification of the <i>ddrA</i> gene deletion by restriction analysis of purified PCR products. Purified PCR fragments were amplified from the genomic DNA of strains R1, TNK104, and TNK110, using primers that flank the coding sequences for <i>ddrA.</i> Products were restricted with EcoR1 (left panel) and EcoRV (right panel) to verify their identity. Products were separated on a 0.8% agarose gel to establish whether the restriction fragment corresponded with the expected sizes as illustrated in the figure above each image of the agarose gel.</p> <p>(C) Verification of the <i>recA</i> gene deletion by restriction analysis of purified PCR products. Purified PCR fragments were amplified from the genomic DNA of strains R1, TNK106, and TNK110, using primers that flank the coding sequences for <i>recA.</i> Products were restricted with PvuII (left panel) and BglII (right panel) to verify their identity. Products were separated on a 0.8% agarose gel to establish whether the restriction fragment corresponded with expected sizes as illustrated in the figure above each gel.</p>