Southern Blot Analysis of CAN1 Gene

For each Southern blot, 3–5 μg of genomic DNA digested with Eco RV and Ssp I were loaded on a 1% agarose gel and electrophoresis was performed overnight at 1V/cm. The gel was manually transferred overnight in 20X SSC, on a Hybond-XL nylon membrane (GE Healthcare), according to manufacturer recommendations. Hybridization was performed with a 302 bp ³²P-randomly labeled CAN1 probe amplified from primers CAN133 and CAN135 (Supplemental Table S2) (22 (link)). Each probe was purified on a G50 column (ProbeQuant G50 microcolumn, GE Healthcare) and specific activities were verified to be above 2.4 × 10⁸ cpm/μg. The membrane was exposed 3 days on a phosphor screen and quantifications were performed on a FujiFilm FLA-9000 phosphorimager, using the Multi Gauge (v. 3.0) software. Percentages of DSB and recombinant molecules were calculated as the amount of each corresponding band divided by the total amount of signal in the lane, after background subtraction.