Summary: Yeast Permanents
1. Prepare large patches on appropriate selective plate. Incubate at appropriate temperature, usually
30°C, until significant growth is visible. Minimal medium usually requires 3-4 days.
Alternatively, yeast from a selective plate can be grown in 3-5 ml of YPD overnight (i.e., grow for
only a limited number of doublings in the absence of selection before making permanents).
2. For patch, collect cells with disposable plastic loop and transfer to 1.5 ml of ice-cold YPD + 25%
(wt/vol) glycerol in labeled cryovial on ice. Do in duplicate.
For cells grown in YPD, centrifuge at 2000 rpm for 5 minutes at 4°C. Resuspend pellet in 3 ml of
ice-cold YPD + 25% (wt/vol) glycerol. Transfer 1.5 ml to two labeled cryovials on ice.
3. Cover cryovials in powdered dry ice (no solvent) for 15 minutes.
4. Place duplicate vials in separate stock boxes in two different -70°C freezers. Note strain name on
list of permanents in lab "red" book.
Recovery of Permanents
Place stock vial in small Stratacooler to prevent thawing. Scrape cells from frozen stock vial with
sterile toothpick or loop. Place on YPD agar medium and grow at appropriate temperature 1-2
days. Never allow frozen stock vial to thaw. Immediately return vial to freezer.
E. coli Permanents
1. Streak bacteria to single colonies on selective plate, usually LB + antibiotic. Grow at appropriate
temperature, usually 37°C.
2. Pick a single colony and inoculate 5 ml of selective liquid medium, usually LB + antibiotic. Grow