Southern Blot Analysis of Genomic DNA

Genomic DNA was isolated from mouse tail in DNA digestion buffer (10 mM Tris pH 8.0, 5 mM EDTA, 0.1 M NaCl, 1% SDS) containing 100 μg proteinase K overnight at 56 °C followed by phenol-chloroform extraction, ethanol precipitation and re-suspension in TE buffer. Genomic DNA was digested overnight with 100 units of NcoI-HF (New England Biolabs) at 37 °C, separated on a 0.8% agarose gel and transferred to a positively charged nylon membrane (Roche). Membranes were hybridized with digoxigenin (DIG)-labelled internal probes (Roche) and visualized by chemiluminescence using an AP-anti-DIG antibody (Roche)^{81 (link)} and CDP-Star (Roche). Membranes were stripped using 0.2 M sodium hydroxide with 1% SDS at 37 °C, rehybridized with DIG-labelled external probes and visualized using the same chemiluminescence protocol. DIG-labelled probes were generated by PCR using the DIG probe synthesis kit (Roche) according to manufacturer’s instructions (see Table S4 for primer sequences).