Random primer dna labeling kit

DNA extraction was performed on cell pellets collected from serial liquid growth cultures at various stages of senescence and therefore required 1–5 mL of overnight culture depending on the genotype. Five independent clones were selected after tetrad dissection and independently processed for serial dilution analysis. At approximately 25 and 80 generations, cells were pelleted and DNA extracted for Southern blot. Purified DNA was digested by XhoI. Following digestion, 10 μg of DNA was separated overnight on a 0.8% agarose gel with a 0.5% TBE buffer system overnight. The DNA was transferred onto nitrocellulose using capillary action and blotted with probes to the Y’-element (Fig 3B). The probes for Southern analysis were prepared using PCR with primers T3 (5’-AGC GCG CAA TTA ACC CTC ACT AAA G-3’) and T7 (5’-CGT AAT ACG ACT CAC TAT AGG G-3’) with pRS313/Y’RsaI template, kindly provided by Dr. Gottschling [106 (link)]. PCR products were radiolabeled with [α-³²P] dCTP using the Random Primer DNA Labeling Kit (Roche). Radiolabeled signal was captured and visualized with the Storm860 PhosphorImager (Molecular Dynamics). Images were processed using ImageQuant TL software, v. 5.2 (Amersham).

Total RNA was isolated and analyzed by northern blotting, as described previously [9 (link)]. The random hexamer probes used to detect hns mRNA were synthesised using a random primer DNA labeling kit (Roche Diagnostics). PCR products containing the hns ORF were synthesised using RT-St-hns-F (+149) and St-hns (stop)-R primers and used as a template to synthesise random hexamer probes. The oligonucleotide probe M1-R was used to probe M1.