Southern Blot Analysis of Rosa26 Locus

Southern blotting for correct integration of the targeting construct into the Rosa26 locus was done as described [24 (link)]. Briefly, genomic DNA was isolated from tails and 10 μg were digested with EcoRI. DNA fragments were separated on a 0.7 % agarose gel. The gel was washed two times for 5 min in dH₂O, incubated two times for 15 min in 0.125 M HCl for depurination, washed two times for 5 min in H₂O, and was finally denatured by incubating two times for 15 min in 0.5 M NaOH/1.5 M NaCl. DNA was blotted over night to a Hybond XL membrane (GE Healthcare). The membrane was then neutralized for 10 min in 0.5 M Tris–HCl pH 7.2/1 M NaCl, dried, and UV-crosslinked by irradiation with 120,000 μJ cm⁻². Then, the membrane was pre-incubated for 3 h at 65 °C in ExpressHyb Hybridization Solution (Takara). 25 ng probe was radioactively labeled using Ladderman Labeling Kit (Takara) by adding 25 μCi ³²P-dCTP and purified on a Sephadex-G50 column (GE Healthcare). The probe was first denatured and then incubated with the membrane over night at 65 °C. Next, the membrane was briefly washed twice in 2 × SSC/1 % SDS, then incubated in 2 × SSC / 1 % SDS for 30 min, then incubated in 1 × SSC / 1 % SDS for 30 min, followed by 0.5 × SSC / 1 % SDS for 30 min, all at 65 °C. The membrane was used to expose an X-ray film at −80 °C in the dark for 3 to 7 days.