Mice were routinely genotyped by Southern blotting restriction digested tail DNA. Tail DNA was prepared using standard procedures [67]. ∼10 µg of DNA was digested with the appropriate restriction enzyme and resolved by 0.5% agarose gel electrophoresis for standard electrophoresis or 1.0% agarose using FIGE. DNA was transferred to Hybond XL (Amersham) using alkaline capillary transfer crosslinked with UV (Stratalinker) and probed with random hexamer labeled probes at 65°C using Perfect Hyb Hybridization Buffer (Sigma). Probes used for blotting were PCR amplified from pCH110 (Genbank # UO2445) using primers JL510 and JL511 for Probe B, and from mouse genomic DNA using primers JL299 and JL300 for Probe A; JL60 and JL61 for probe exon2; JL235 and JL236 for probe 29–31; JL293 and JL294 for probe 25–26; JL295 and JL296 for probe 20.2–20.7 using Taq polymerase (Invitrogen) (see Table S3 for primer sequences). Blots were washed twice with 2.0× SSC, 0.1% SDS and twice with 0.5× SSC, 0.1% SDS for 10 min @ 65°C for each wash. A Phosphoimager was used to detect the hybridization signal.
