101 148 063 microtip

Genomic DNA was isolated by incubating cells in SDS-PK buffer (0.5% (wt/vol) sodium dodecyl sulfate (SDS), 50 mM Tris-HCL, 0.01M EDTA, 1M NaCl and 0.2 mg/ml proteinase K) at 56 °C for 2 h. Phenol-chloroform extraction was performed and genomic DNA was precipitated in 1 volume of isopropanol at -20 °C. DNA was pelleted, washed with ethanol and resuspended in TE buffer. Genomic DNA was fragmented to an average size of 200 bp using a Branson Digital Sonifier (Model 250) and a Branson 101-148-063 microtip. DNA was sonicated using 40% amplitude for a total of 4 minutes. A double-antibody immunoprecipitation was performed to isolate nascent (BrdU labeled) strands of DNA. For each immunoprecipitation reaction, at least 120 ng of sonicated DNA was used. DNA was first heat denatured at 95 °C for 5 min and rapidly cooled on ice. 12.5 μg primary antibody (mouse anti-BrdU, BD Biosciences Pharmingen, cat. no. 555627) was added to the ssDNA suspension with constant rocking for 20 minutes followed by addition of 20 μg of secondary antibody (rabbit anti-mouse IgG, Sigma, cat. no. M-7023) for 20 min. Antibody-DNA complexes were then pelleted, resuspended and incubated in digestion buffer (50mM Tris-HCl, 0.01M EDTA, 0.5% SDS and 0.25 mg/ml proteinase K) at 37 °C overnight. Nascent ssDNA was subsequently purified by phenol-chloroform extraction and ethanol precipitation.