Click-iT 5-ethynyl-2′-deoxyuridine immunoprecipitation

293T cells were transfected with indicated plasmids. Forty-eight hours after transfection, cells were labeled with 30 μM EdU (5-ethynyl-2′-deoxyuridine) for 40 min and then harvested for total DNA extraction using Hirt’s method. DNA was then sonicated to get an average length of 500 to 750 bp. Click-iT reaction was then performed by addition of Click-iT reaction components and incubation for 2 hours at room temperature. Increasing the sample volume to 500 μl, added streptavidin beads and the samples were rotated at room temperature for overnight. Next day, samples were washed twice with washing solution [10 Mm tris-HCL (pH 7.5), 1 mM EDTA, and 2 M NaCl] and once in distilled water. The samples were then eluted by resuspending beads in elution buffer (1% SDS and 0.1 M NaHCO3) and incubating at 65°C for 10 min, followed by centrifugation and transfer of supernatants to new tubes. DNA was precipitated with a DNA purification kit from TIANGEN and resuspended in 50 μl TE. This DNA was next used in qPCR analysis to assess levels of replicating DNA at cellular c-Myc replication origins.

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