Abstract
Here we describe a protocol for the generation of site-specific DNA damage, including double and single strand breaks, using the 405 nm laser of a confocal microscope in cells pre-sensitized with Hoechst. This is a simple approach, particularly useful to assess the involvement of proteins and the roles of liquid-liquid phase separation in DNA damage repair. Examples of transfection protocol, drug concentrations, and microscopy are provided, although optimization may be needed for specific experimental setups and cell lines used. For complete details on the use and execution of this protocol, please refer to Levone et al. (2021).