This collection of protocols describes the preparation of reagents for transfecting DNA into the choanoflagellate Salpingoeca rosetta using a Nucleofector 4d Device and SF kit from Lonza. Plasmids that use S. rosetta promoters and regulatory elements to drive the expression of luciferase and fluorescent protein reporters are being deposited at Addgene. Acknowlegements: Arielle Woznica and Laura Wetzel for meticulous editing of protocol and clarifying steps in the procedure. The Gordon and Betty Moore Foundation for supporting this research.