Protocol for analyzing DNA dynamics in the presence of crowders and confined within cell-sized droplets

Here, we present a protocol for encapsulating DNA molecules under crowded conditions within cell-sized lipid-coated droplets. We describe steps for preparing a lipid-oil mixture and adding an aqueous solution containing DNA, which, when mixed, forms water-in-oil droplets of radii between ∼5 and 100 microm. We then detail procedures for quantifying the dynamics of DNA molecules in these droplets by analyzing fluorescence microscopy time series using differential dynamic microscopy. This protocol can be utilized to investigate DNA transport within a range of biomimetic and crowded environments. For complete details on the use and execution of this protocol, please refer to Aporvari et al.¹.