Chef drii pfge apparatus

At the indicated time points, 1 × 10⁶ cells were harvested by trypsinization, suspended in PBS, mixed with 2% agarose (1:1 ratio), and casted in a plug mold. Plugs were digested overnight in proteinase K digestion buffer (10 mM Tris-HCl pH 8.0, 250 mM EDTA, 0.2% sodium deoxycholate, and 1% sodium lauryl sarcosine) at 55°C. After extensive washes with Tris EDTA (TE), plugs were incubated with 60 U MboI overnight at 37°C. Digested DNA was resolved on a 1% agarose/0.5XTBE gel using a CHEF-DRII PFGE apparatus (Bio-Rad) for 24 hr. The gels were then dried at room temperature and hybridized overnight at 50°C with γ-³²P-ATP end-labeled (AACCCT)₄ probe in Church mix (0.5 M sodium phosphate, pH 7.2, 1 mM EDTA, 0.7% SDS, and 0.1% BSA). The gel was washed at 50°C three times in 4XSSC (30 min each), once in 4XSSC/0.1% SDS (30 min), and exposed to a PhosphoImager screen. After capturing the single-stranded telomere signal, the DNA was denatured in situ with 0.5 M NaOH/1.5 M NaCl for 30 min, neutralized with two 30 min washes in 0.5 M Tris-HCl pH 7.5/3 M NaCl, prehybridized in Church mix for 30 min at 55°C, and hybridized overnight with the same probe at 55°C. The next day, the denatured gel was washed as described above and exposed to capture the total telomere signal. Signals were quantified using ImageJ.