Abstract
Cell-free protein synthesis (CFPS) is an established biotechnology tool that has shown great utility in many applications such as prototyping proteins, building genetic circuits, designing biosensors, and expressing cytotoxic proteins. Although CFPS has been widely deployed, the many, varied methods presented in the literature can be challenging for new users to adopt. From our experience and others who newly enter the field, one of the most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present within experimental replicates. Herein we provide a retrospective summary of CFPS methods that reduce variability significantly. These methods include optimized extract preparation, fully solubilizing the master mix components, and careful mixing of the reaction. These have reduced our coefficient of variation from 97.3% to 1.2%. Moreover, these methods allow complete novices (e.g. semester rotation undergraduate students) to provide data that is comparable to experienced users, thus allowing broader participation in this exciting research area.