Yeast colony cryosections:
1) Cut a square of agar surrounding a 2-3 mm colony from an agar plate and submerge it in 1.25 ml of OCT compound (VWR 25608-930) in a 15 X 15 X 5 mm cryomold (VWR 25608-924).
2) Cover this cryomold with another cryomold to prevent the OCT from drying, and incubate at room temperature for 90 min.
3) With 20 minutes to go before the next step, submerge a 200 ml tin can (e.g. tomato sauce) filled with isopentane in a Styrofoam box containing liquid nitrogen. The nitrogen should cover the can to the outer rim, during the course of the protocol it is usually necessary to continue to top up the liquid nitrogen.
3) After the 90 min incubation, cryomolds containing the sample are submerged for 20 sec in the isopentane using forceps. The colonies can then be stored in -80°C freezer and can be transported to the cryostat on dry ice.
4) Colonies are sectioned at -20°C ---25°C using a Microm HM 505E cryostat. 20 um sections through the central region of the colony are covered with mounting media (KPL 71-00-16) and a cover slip, sealed with nail polish and examined using Nomarski optics.
