Annealing and purification of fluorescently labeled DNA substrates for in vitro assays

We present a protocol to generate high-quality fluorescently labeled DNA substrates that can be used for biochemical assays, including DNA-binding and nuclease activity assays. We describe polyacrylamide-gel-electrophoresis-based purification of DNA oligonucleotides, followed by annealing the oligonucleotides and purifying the annealed substrates using anion-exchange chromatography. This protocol circumvents the use of radioisotopes, which require training and dedicated equipment for safe handling and necessitate specialized waste disposal. This protocol is amenable to varying lengths of oligonucleotides and DNA substrates. For complete details on the use and execution of this protocol, please refer to Payliss and Tse et al. (2022).¹.