Single-molecule imaging studies using long linear DNA substrates have revealed unanticipated insights into the dynamics of multi-protein systems. The use of long DNA substrates allows for the study of protein���DNA interactions with observation of the movement and behavior of proteins over distances accessible by fluorescence microscopy. Generalized methods can be exploited to generate and optimize a variety of linear DNA substrates with plasmid DNA as a simple starting point using standard biochemical techniques. Here, we present protocols to produce high-quality plasmid-based 36-kb linear DNA substrates that support DNA replication by the Escherichia coli replisome and that contain chemical lesions at well-defined positions. These substrates can be used to visualize replisome���lesion encounters at the single-molecule level, providing mechanistic details of replisome stalling and dynamics occurring during replication rescue and restart.