Intron Insertion Detection in C. perfringens

DNA was extracted from all C. perfringens strains using the MasterPure Gram-positive DNA purification kit. PCR for the virS or agrB genes was performed using the primers described in the previous section. For the wild-type strains, the sizes of PCR products are listed in Table 4. For the null mutant strains, PCR using the same pair of the primers should amplify a product from the intron-disrupted gene that is ∼900 bp larger than the gene from the wild-type strains. All PCR conditions were described previously (25 (link), 30 (link)).
For Southern blot analysis to detect an intron insertion, aliquots (3 μg each) of wild-type, single-null, or double-null mutant strain DNA were digested overnight with EcoRI at 37°C according to the manufacturer’s instructions (New England Biolabs). The digested DNA samples were then electrophoresed on a 1% agarose gel before transfer onto a positively charged nylon membrane (Roche) for hybridization with an intron-specific probe (20 (link)). The intron-specific probe was prepared using the PCR digoxigenin (DIG) probe synthesis kit (Roche) and intron primers (IBS and EBS2). After hybridization, Southern blots were developed using reagents from the DIG DNA labeling and detection kit (Roche) according to the manufacturer’s instructions.