Southern blots were performed according to [41 (link)] and carried out on 5 μg of P. caerulea DNA digested with 10 units of HaeIII at 0, 5, and 15 min; 1 h; and overnight.
For the other investigated molluscan species (P. depressa, P. vulgata, P. rustica, P. ferruginea, P. ulyssiponensis), Southern blot was performed on 5 μg of DNA digested overnight with HaeIII. Hybridization was carried out as described by [41 (link)] using the biotinylated clone 2 of PcH-sat. Washing was performed at medium stringency conditions [2 (link)] washes in 0.2× standard saline citrate solution (SSC) and 0.5% sodium dodecyl sulphate (SDS) for 15 min. at 55 °C. Hybridization signals were revealed by 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (BCIP-NBT) (Promega, Madison, WI, USA).
For the other investigated molluscan species (P. depressa, P. vulgata, P. rustica, P. ferruginea, P. ulyssiponensis), Southern blot was performed on 5 μg of DNA digested overnight with HaeIII. Hybridization was carried out as described by [41 (link)] using the biotinylated clone 2 of PcH-sat. Washing was performed at medium stringency conditions [2 (link)] washes in 0.2× standard saline citrate solution (SSC) and 0.5% sodium dodecyl sulphate (SDS) for 15 min. at 55 °C. Hybridization signals were revealed by 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (BCIP-NBT) (Promega, Madison, WI, USA).