Establishment of cohesion between sister chromatids in mitosis is one of the fundamental principles used by cells to ensure accuracy of the chromosome segregation. Cohesion allows spindle microtubules to generate tension across sister chromatids that acts as a signal that correct spindle-chromosome connections have been made. Cohesion is established by cohesin—a ring-shaped protein complex that is thought to link sister chromatids by physically entrapping their DNA. However, molecular mechanisms underlying cohesin interaction with DNA and its ability to form DNA–DNA interactions are not well understood. Here, we describe a protocol to reconstitute this process at the single-molecule level and investigate how single cohesin protein complexes establish DNA–DNA interactions and resist mechanical forces exerted on DNA.