Analyzing DNA Replication Using Southern Blotting

Total intracellular DNA was extracted from SV40- and mock-infected cells and subjected to agarose gel electrophoresis and Southern blotting as described [30] (link). In Figure 1, the same amount of SV40 DNA was loaded into each lane. In all other Southern blots, loading was normalized to cell number. In both cases, mitochondrial DNA was probed as an internal loading control. Loading determined by cell number or by mitochondrial DNA signal was highly correlated. 2D gel electrophoresis was as described by [30] (link) with the following modification: the second dimension of the 2D gel was run for 7 h through a 0.95% 1×TBE agarose gel containing 0.5 ng/mL ethidium bromide. Southern blotting probes and data analysis using ImageQuant 5.2 were as previously described [30] (link).
For DNA extractions from cells exposed to 20 µM EdU for 30 minutes (Figure 6A), DNA was isolated as per [30] (link), but dissolved in 1 µL of 10 mM Tris pH 8.0 with 0.1 mM EDTA per 20,000 cells. A rat anti-BrdU antibody ([BU1/75 (ICR1)], Abcam), anti-rat conjugated to HRP (Jackson ImmunoResearch), and ECL-plus reagent (Perkin Elmer) were used to detect EdU label on the Southern blot as described in [83] (link).