Many of these steps will be performed on the same day - excepting the preparation of stable stock solutions and image analysis.
We recommend starting with the buffer-in-buffer liposome protocol. This protocol is intended to familiarize you with the mechanics of assembling liposomes and to help you develop your characterization and analysis pipelines. These protocols can be performed in a day or two and require very few specialized components.
After getting comfortable with these protocols, we recommend building PURE liposomes to run a few basic modules, such as the reporter module GFP to test protein expression or the pore-forming module pT7-aHly to enable the exchange of small molecules between the environment and your synthetic cells (Alpha hemolysin). These protocols will require several specialized components that may take some time to assemble. Additionally, care should be taken to plan out your PURE reactions before entering the lab.
After preparing your liposomes, microscopy is simplest means to characterize them. Since microscopy will be highly specific to your specific circumstances, we currently provide some guidelines for sample preparation and imaging, tips for troubleshooting, and details to consider recording when sharing your data. We also provide an image analysis toolkit that will be useful for evaluating data obtained under a variety of imaging conditions. These resources are described here:
