3D positioning of tagged DNA loci by widefield and super-resolution fluorescence imaging of fixed yeast nuclei

Abstract

Summary: This protocol describes how to culture, image, and determine the nuclear position of a fluorescently tagged DNA locus in the 3D nucleoplasm of fixed Saccharomyces cerevisiae cells. Here, we propose a manual scoring method based on widefield images and an automated method based on 3D-SIM images. Yeast culture conditions have to be followed meticulously to get the best biological response in a given environment.For complete details on the use and execution of this protocol, please refer to Forey et al. (2020).

Keywords

• Cell culture
• Single Cell
• Genetics
• Microscopy
• Model Organisms
• Gene Expression