Regulatory mechanisms for DNA replication are crucial to the control of the cell cycle in eukaryotic cells. One of the widely used assays to understand the complex mammalian replication system is the cell-free in vitro replication assay (IVRA). IVRA can provide a snapshot of the regulatory mechanisms controlling replication in higher eukaryotes by using a single plasmid, pEPI-1. This chapter outlines the general strategies and protocols used to perform IVRA to study the differential recruitment of replication factors either independently or in combination, based on the experience in studying the role of prohibitin in replication as well as other published protocols. This method can be employed to identify not only proteins that assist replication but also proteins that inhibit replication of mammalian genome.
