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Summary: Here we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel. Finally, we detail how to quantify the gel images.For complete details on the use and execution of this protocol, please refer to Appling et al. (2015).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.