FIGURE 2.1 - Preparing X/I/C trays. The work surface of the laminar-flow hood should be thoroughly wiped clean with 70% ethanol before starting. A Bunsen burner should be set up in the hood. Due to the light-sensitivity of X-GAL, the lights in the laminar-flow hood should be turned off. Room lights also should be dimmed. (a) A 250 ml beaker is filled to exactly 200 ml with X/I/C media. Any bubbles in the media are eliminated by quickly passing the flame of the Bunsen burner over the surface of the media. (b) The lid is removed from a UV-sterilized 500 cm² tray, and the contents of the beaker are poured into the tray. (c) The lid is rotated 45° with respect to the tray and placed on top of the tray. (d) The first tray is used as a base on which a second tray is prepared, the second tray as a base for preparation of a third tray, etc. Ten to twenty plates can be stacked in a column. After all trays have been filled with 200 ml of X/I/C, allow the plates to sit undisturbed in the dark for 45 minutes. During this time the media in the plates should solidify. (e) Replace the tray lids, and invert each tray. (f) Inverted trays can be stacked as shown. Preferably, X/I/C trays should be plated with transformants the same day they are made. However, trays can be stored in darkness at room temperature for 1-4 days or wrapped in plastic wrap and stored in the dark at 4°C for several weeks. If extra X/I/C agar is left after preparing trays, use the remaining media to make X/I/C Petri plates.