Quantifying DNA Replication Dynamics

For detection of replication activities, each 10 μl extract sample was incubated with 2 μM of Cy5-dUTP (GE Healthcare) for the appropriate number of times and was diluted with 200 μl of stop buffer (5-mM EDTA, 200-mM NaCl, 0.5% SDS, 20-mM Tris-HCl, pH 8.0) containing 200 μg/ml Proteinase K (Roche) and 10 μg/ml RNaseA (Sigma-Aldrich) and incubated for 2 h at 37 °C. The genomic DNA was purified by phenol/chloroform extraction and ethanol precipitation and electrophoretically separated by 0.8% tris-acetate EDTA (TAE) agarose gel (neutral condition) or 1% alkaline agarose gel (denaturing condition) as described (50 (link)) and stained with SYBR Gold (Invitrogen). The alkaline gel was neutralized with PBS before SYBR Gold staining because it is pH sensitive. The signals of incorporated Cy5 (Replication activity) and SYBR Gold (total genomic DNA) were scanned with Typhoon FLA 9000 Gel Imager (GE Healthcare) and quantified with ImageJ software.