Pulse-Chase Labeling of Yeast with
[3H]-Methyl Methionine or [3H]-Uracil
· Plan for: 0.5 A600 unit of cells per chase time,
0.5 ml of labeling medium per A600 unit,
25 µCi of radioactive label per A600 unit,
5 µl of volume to resuspend RNA pellet from 1 A600 unit of cells (enough for 2 gels).
· For [3H]methyl methionine labeling, use methionine-free medium.
· For uracil labeling of uracil auxotrophs, use 1/3 the normal amount of uracil supplement.
Medium or Temperature Shift
1. Prepare a starter culture of log phase yeast (A600 0.5). For a standard depletion experiment,
grow in SGal plus supplements. For a standard temperature shift experiment, use SD plus
supplements. Plan for both experimental and control cell cultures.
2. Medium Shift: If you plan to change the medium, wash once in sterile ddH2O at room temperature.
Spin down cells at 2000 rpm for 5 minutes in IEC centrifuge at 25°C.
Temperature Shift. If you plan to raise the temperature to 37°C, add 50°C medium to 37°C, and
transfer to incubator-shaker pre-warmed to 37°C.
3. Plan to collect a total of 2.5 A600 units of cells at A600 < 0.5 for the pulse-chase labeling. For this,
dilute cells into the appropriate medium such that you will have enough cells at the desired time in
the desired medium at the desired temperature. Plan to grow excess culture volume (5-10 A600