The ability to apply controlled forces to individual molecules or molecular complexes and observe their behaviors has led to many important discoveries in biology. Instruments capable of probing single-molecule forces typically cost >US$100,000, limiting the use of these techniques. The centrifuge force microscope (CFM) is a low-cost and easy-to-use instrument that enables high-throughput single-molecule studies. By combining the imaging capabilities of a microscope with the force application of a centrifuge, the CFM enables the simultaneous probing of hundreds to thousands of single-molecule interactions using tethered particles. Here we present a comprehensive set of instructions for building a CFM module that fits within a commercial benchtop centrifuge. The CFM module uses a 3D-printed housing, relies on off-the-shelf optical and electrical components, and can be built for less than US$1,000 in about 1 day. We also provide detailed instructions for setting up and running an experiment to measure force-dependent shearing of a short DNA duplex, as well as the software for CFM control and data analysis. The protocol is suitable for users with basic experience in analytical biochemistry and biophysics. The protocol enables the use of CFM-based experiments and may facilitate access to the single-molecule research field.
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