DNA Fiber Analysis: Quantifying Replication

DNA fibers were analysed using a protocol previously used by us (Mansilla et al., 2013 (link)) with a minor change in the time of labelling. Exponentially growing cells were pulse labeled with CldU (20 µM) for 10 min, washed twice,and incubated with IdU(200 µM) for additional 30 min. Cells were trypsinized and lysed with 6 µl of 0.5% SDS, 200 mM Tris–HCL (pH 7.4) and 50 mM EDTA buffer onto clean glass slides, which were tilted, allowing DNA to unwind. Samples were fixed in 3:1 methanol/acetic acid and denatured with HCL (2.5 N) for 1 h, blocked in PBS 5%Bovine serum albumin (BSA) for 15 min and incubated with the mouse anti-BrdU (Becton Dickinson, USA) to detect IdU, donkey anti-mouse Cy3-conjugated secondary antibody (Jackson Immuno Research, West Grove, Pennsylvania), rat anti-BrdU (Accurate Chemicals, Westbury, New York) to detect CldU and donkey antirat Alexa 488 secondary antibody (Invitrogen). Slides were mounted with Mowiol 488 (Calbiochem), and DNA fibers were visualized using a Zeiss Axioplanconfocal microscope. Images were analysed using Zeiss LSM Image Browser software and Image J software. Each data set is derived from measurement of 85–100 fibers.