Genomic DNA was isolated from purified aged populations by standard methods. 1 µg of genomic DNA was digested overnight with BamHI + RecBCD (a gift from G. Smith, FHCRC) and separated by gel electrophoresis (0.8% agarose, 2 V/cm for 36 hours). DNA was visualized by staining with ethidium bromide and transferred to nitrocellulose membranes by standard methods. Membranes were hybridized with a 32P labeled double-stranded probe specific to the rDNA generated with oligos RDN5S-2 and RDN25S-2 using plasmid pDL05 as a template, visualized on a Typhoon phosphorimager (GE Health Sciences), and quantified using ImageJ. For normalization of DNA samples, 1 µg of genomic DNA was digested with BamHI, separated by gel electrophoresis (0.8% agarose, 10 V/cm, 3 hours), transferred and hybridized with a probe specific to NPR2 generated with oligos 5_NPR2 and 3_NPR2.
