Anti s9

Genomic DNA was prepared as above. 5 μg DNA was digested with 3 μL Hind III (Fisher, FD0504), 3 μL EcoRI (Fisher, FD0274), and 3 μL Bam HI (Fisher, FD0054) +/−5 μL RNase H (NEB, M0297) in RNase H buffer (NEB, M0297) overnight at 37°C. Digested DNA was incubated with 50 μL of protein A/G agarose beads (Santa Cruz, sc-2003) and 10 ul mouse serum (MP Biomedicals, 152282) in 1.5 mL PBS/0.5% Triton X100 for 2 h at 4°C. Digested DNA was then incubated with 50 μL of protein A/G agarose beads and 5 μL anti-S9.6 (Millipore, MABE1095) overnight at 4°C. Agarose beads were washed 3× for 3 min in PBS/0.5% Triton X100 binding buffer followed by PBS wash for 3 min. The beads were then incubated with 3 μL proteinase K for 2 h at 37°C. Nucleic acids were purified by phenol/chloroform extraction and ethanol precipitation. DNA was denatured in 50 μL TE and mixed with 50 μL of 0.8 M NaOH/20 mM EDTA solution for 10 min at 95°C then placed on ice. The solution was neutralized using sodium acetate pH 7.0 and DNA sample was transferred to Whatman Hybond N+ Blotting Membrane (Millipore Sigma, Z761079). Nucleic acids were cross-linked by to the membrane by baking at 60°C for 30 min followed by UV exposure. The membrane was blocked for 1 h with 5% milk in TBST and blotted for anti-ssDNA (1:1000, Millipore, MAB3034).