Rpn119b

A total of 50 ng ChIPed DNA was denatured incubating for 10 min at 99°C with 0.4 N NaOH, and 10 mM EDTA. Denatured DNA samples were applied to hybond N⁺ (Amersham; RPN119B) membrane using a dot-blot apparatus connected to a vacuum source and washed with 2× SSC once. DNA was denatured on a membrane using 1.5 M NaCl/0.5 N NaOH for 10 min and then neutralized using 1.5 N NaCl/0.5 M Tris pH 7. DNA was UV crosslinked using a UV Stratagene crosslinker. Membrane was prehybridized by rapid hybridization buffer (Amersham; RPN1635) for 1 h and incubated over night with radiolabeled telomere (TTAGGG)₃ or Alu probe (5′-CGGGAAGCAGAGGTTGTAGTGAGCC). Reaction mixture was 1 μl telomere restriction fragment, 13.5 μl H₂O, 2 μl T4 PNK buffer, 2.5 μl ³²P-γATP and 1 μl T4-polynucleotide kinase (New England Biolabs) at 37°C. The membrane was washed the next day once with 0.1% SDS/2× SSC buffer and twice with 0.1% SDS/1× SSC. Radiolabeled membranes were exposed to a phosphorimager (Fuji), and signal intensities were quantified after 3 days exposure.