Egg Incubation and Collection of Painted Turtle Embryos Nicole Valenzuela Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, IA 50011, USA INTRODUCTION This protocol describes how to incubate painted turtle eggs and collect embryos for gene expression analysis. The same basic protocol can be used to incubate eggs and collect embryos from other tur- tle species by modifying the incubation temperature to suit the particular developmental needs of each species. RELATEDINFORMATION A companion article, The Painted Turtle, Chrysemys picta: A Model System for Vertebrate Evolution,Ecology,andHumanHealth (Valenzuela 2009) describes how the painted turtle is becom- ing an emerging vertebrate model for ecological, physiological, evolutionary, and genomic studies. © 2009 Cold Spring Harbor Laboratory Press 1 Vol. 4, Issue 7, July 2009 Corresponding author (nvalenzu@iastate.edu) Cite as: Cold Spring Harb Protoc; 2009; doi:10.1101/pdb.prot5238 www.cshprotocols.org Protocol MATERIALS CAUTIONSANDRECIPES: Please see Appendices for appropriate handling of materials marked with <!>,and recipes for reagents marked with <R>. Reagents Chrysemys picta eggs Eggs should be as fresh as possible; eggs ovoposited the night before are best. Incubation substrate (fine play sand or medium grade vermiculite) RNAlater (Applied Biosystems) RNAse-away (Sigma-Aldrich) or equivalent surface decontaminant Equipment Balance (≥3000g capacity and 1 g readability or better) Dissecting tools (fine scissors and fine forceps; spatula for smaller embryos as described in Step 18) Incubation boxes (plastic boxes with lids) Incubators (set to desired temperatures, see Step 7; ±0.5°C accuracy or better) <!>Liquid nitrogen (optional; see Step 23) Pencil (No. 2) or indelible ink pen (e.g., Sharpie) Petri dishes Squirt bottle Vials for embryo collection (2-, 10-, 15-, and 50-mL, depending on size of embryos and dissected tissues)