Magnetic tweezers provide a versatile tool enabling the application of force and torque on individual biomolecules. Magnetic tweezers are uniquely suited to the study of DNA topology and protein-DNA interactions that modify DNA topology. Perhaps due to its presumed simplicity, magnetic tweezers instrumentation has been described in less detail than comparable techniques. Here, we provide a comprehensive description and guide for the design and implementation of a magnetic tweezers instrument for single-molecule measurements of DNA topology and mechanics. We elucidate magnetic trap design, as well as microscope and illumination setup, and provide a simple LabVIEW-based real-time position tracking algorithm. In addition, we provide procedures for production of supercoilable DNA tethers, flow-cell design, and construction tips.