From Powers Lab UC Davis
1. Inoculate 5 mL of YPD with toothpick
swig of yeast strain of interest (grown from frozen permanent stock) and grow
at 30°C for several hours.
2. Inoculate 1 L of YPD with aliquot
of overday culture (amount is based on spectrophotometric reading of overday
culture and calculation of approximation of time it will take for overnight
culture to reach a certain O.D.600 ).
3. Grow cells to O.D.600 between
0.5 and 1.0.
4. Divide culture equally between
4 x 250 mL Corning disposable centrifuge tubes.
5. Centrifuge in Allegra tabletop
centrifuge at approximately 4000 rpm for 10 min., room temperature.
6. Discard supernatant.
7. Resuspend each pellet (4 of them)
in 50 mL cold sterile-filtered H2O. Use a 25 mL disposable pipet and a Pipet-Aid
to gently resuspend the cells.
8. Consolidate resuspended cells/H2O
to only 2 of the 250 mL Corning tubes.
9. Centrifuge in Allegra tabletop
centrifuge at approximately 4000 rpm for 10 min, at room temperature.
10. Discard supernantant.
11. Resuspend each of the two pellets
in 50 mL of cold 1X Yeast Extract Buffer (see Basic Recipe below). Transfer
the cells/yeast extract buffer into 2 x 50 mL blue-capped Falcon tubes.
12. Centrifuge in Allegra tabletop
centrifuge at approximately 4000 rpm for 10 min., at room temperature.
13. While cells are spinning, get
liquid nitrogen and pour some into a 50 mL blue-capped Falcon tube (to about
half to two-thirds full). Liquid nitrogen will boil at first. Poke three or
four holes in the blue cap with a needle.
14. Discard supernatant.
15. Resuspend cells in cold 1X Yeast
Extract Buffer containing protease inhibitors and phosphatase inhibitors. Volume
of buffer used to resuspend the cells should be equal to the volume of the cell
pellet (1:1 cell pellet to buffer).
16. Resuspend cells using a disposable
2-mL transfer pipet and then immediately drip the cell/buffer slurry into the
liquid nitrogen in the 50 mL blue-capped Falcon tube forming cell/buffer "bullets".
17. Recap the Falcon tube and, as
soon as the liquid nitrogen has boiled off, store at -80°C. Even if there
is a small amount of liquid nitrogen remaining, it is OK to go ahead and put
it in the -80°C freezer, because it will finish boiling off in there.