Telomere Length Quantification by Southern Blot

Isolated DNA (3 μg of each sample) was digested with different combinations of REs (as described in the figures) and then separated on a 0.7% agarose gel at 2 V/cm for 16 h. The telomere signals were detected by Southern blot analysis as previously indicated^{37 (link)}. In brief, after DNA was transferred from gel to a positive-charged nylon membrane, the transferred DNA was fixed by UV crosslinking. The cross-linked membrane was then hybridized with the hypersensitive DIG-labeled telomere probe overnight at 42 °C. After hybridization, the membrane was washed with buffer 1 (2× SSC, 0.1% SDS) at room temperature for 15 min and then washed twice with buffer 2 (0.5× SSC, 0.1% SDS) at 55 °C for 15 min. Next, the membrane was washed with DIG wash buffer (1× maleic acid buffer with 0.3% Tween-20) for 5 min. Then the membrane was incubated with 1× DIG blocking solution for 30 min at room temperature. After blocking, the membrane was incubated with anti-DIG antibody (Roche) in 1× blocking solution (1 to 10,000 dilution) for 30 min at room temperature. The membrane was then washed with DIG buffer two times at room temperature for 15 min. After washing, telomere signals were detected by incubating with CDP-star for 5 min. Image quantification was performed using Image Quant software to measure the intensity value of each telomere smear. The intensity value of each sample was then adjusted with the background graphed from a lane with no DNA sample⁶⁶ .