Isolation of biotinylated ARS1 origin

DNA templates were isolated from the pARS1-Nco-Nco plasmid (Kang et al., 2014 (link)). The plasmid was digested with BamHI, filled in with biotinylated-dATP, dGTP, dCTP and dTTP using Klenow enzyme (NEB) to facilitate attachment to beads. After spin column purification (Plasmid Miniprep Kit from Qiagen), the biotin labeled DNA was cut with Nsi I and Sac II followed by a second spin column purification. This creates a large 3.8 kb BamHI to Nsi I DNA fragment that is biotinylated at one end and that is entirely derived from native S. cerevisiae sequences surrounding the ARS1 origin of replication. A small biotinylated DNA (released by Sac II) is removed by the spin column and the remaining bacterial/vector DNA is not biotinylated and will not bind to streptavidin beads. The 3.8 kb biotinylated-DNA was immobilized on streptavidin-coated paramagnetic beads (Dynabeads M-280, ThermoFisher) according to manufacturer instructions and the non-biotinylated DNA fragment was washed away.