Unlabeled telomeric probes were generated using the primer dimer extension method described in Ijdo et al. (1991 (link)), but we used PrimeSTAR Max DNA polymerase (Clontech, R045A) instead of Taq polymerase for the PCR step. The purified PCR products were labeled with digoxigenin-11-dUTP, alkali stabile (Roche, 11093088910) using the DecaLabel DNA Labeling Kit (Thermo Scientific, K0621). The labeled probes were purified using columns from the QIAquick Gel Extraction Kit (Qiagen, 28704) and eluted into the final volume of 50 µl.
One liter of M. exilis culture was filtered to remove bacteria and the cells were pelleted by centrifugation for 10 min at 1,200 × g at 4 °C. FISH with digoxigenin-labeled probes was performed according to the previously described procedure (Zubáčová et al. 2011 (link)) except that the culture was not treated with colchicine and the stringency washes were performed at 45 °C. For probe detection, we used DyLight 594 Labeled Anti-Digoxigenin antibody (Vector Laboratories, DI-7594). Preparations were observed using an IX81 microscope (Olympus) equipped with an IX2-UCB camera. Images were processed using Cell-R software (Olympus) and Image J 1.42q. The number of signals from each nucleus was manually counted and the average number of signals was estimated from at least 50 nuclei.
One liter of M. exilis culture was filtered to remove bacteria and the cells were pelleted by centrifugation for 10 min at 1,200 × g at 4 °C. FISH with digoxigenin-labeled probes was performed according to the previously described procedure (Zubáčová et al. 2011 (link)) except that the culture was not treated with colchicine and the stringency washes were performed at 45 °C. For probe detection, we used DyLight 594 Labeled Anti-Digoxigenin antibody (Vector Laboratories, DI-7594). Preparations were observed using an IX81 microscope (Olympus) equipped with an IX2-UCB camera. Images were processed using Cell-R software (Olympus) and Image J 1.42q. The number of signals from each nucleus was manually counted and the average number of signals was estimated from at least 50 nuclei.