Southern Blot Quantification of DNA Repair

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 1 V/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 [36 (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. Note that DSB and repaired values were taken from Poggi et al. [12 (link)] for each strain, except for NR-Cpf1 for which two additional time courses and Southern blots were run.