Southern blotting hybridization

Genomic DNA (15 μg) was digested with EcoRI (20 units) in 150 µl reaction volume, at 37 °C overnight. DNA fragments were separated on a 0.8 % agarose gel electrophoresis overnight, and transferred to nylon membranes (Hybond-N+, Amersham) by capillary transfer according to Sambrook and Russell (2001). DNA fragments were bound to the membrane by UV cross- linking (Southern Stratalinker 2400). cpPLRV gene (279 bp) was used as probe labeled with [α32] dCTP using the Gene Images Random Prime Labeling kit according to the manufacturer’s recommendation. The probes were hybridized with the membranes at 65 °C for 18 h. The hybridized filter was washed with 1x SSC, 0.1 % SDS (w/v) and 0.5 × SSC, 0.1 % SDS (w/v) at 65 °C for 15 min with gentle agitation. The blot was wrapped in a polyethylene sheet and exposed to radiographic film (Kodak). To detect excision events, we used as probe the nptII gene (638 bp) labeled with PCR DIG Synthesis (Roche kit) according to the manufacturer’s conditions. The probe was hybridized at 65 °C for 30 min with 15 ml of DIG Easy Hyb solution (Roche kit) at 25 ng/mL. The membrane was washed using stringent conditions and exposed to X-ray film (Kodak) at room temperature for 16 h.