Joyce: Preparing RNA from yeast

Procedure

Obtaining cells

1. Streak a cell culture onto a plate to obtain single colonies
2. Allow ample time for cells to grow (about 3 days at 30°C)
3. Pick a cell and innoculate 4 ml of YPD solution
4. Allow ample time for cells to grow (about 3 days at 30°C)
5. Take an aliquot and add glycerol to put in the freezer ******
6. Prepare a series of 50 ml sterile falcon tubes containing 12.5 ml of YPD solution
7. Innoculate each with varying volumes of the 4 ml culture in duplicate (1 ul, 2.5 ul, 5 ul, etc.)
8. Let grow overnight with shaking at 30°C
9. Check OD₆₄₀ the following morning for samples between 0.5 - 1.0
10. Pellet the cells, remove YPD
11. Wash with 500 ul sterile water (vortex)
12. Pellet
13. Pipette out water

Preparing spheroblasts

1. Prepare a zymolase solution as follows:

  4 mg zymolase
  990 ul SCE
  10 ul β-mercaptoethanol

2. Add this to the yeast pellet, vortex

3. Incubate at 37°C for 30-60 minutes, occasionally mixing by inverting

4. After 30 minutes, you can check for spheroblast formation (see notes)

5. Spin at 2000 rpm for 10 minutes

6. Remove supernatant and proceed to extract RNA with your favorite kit

Notes

• The absorbance between 0.5-1.0 indicates log phase growth, ideally you'd want something in the 0.9 range??? ********
• To check for spheroblast formation, collect 4 ul of the zymolase/cell solution and add 20 ul of water to 2 ul and 20 ul of SCE to the other 2 ul. Now that the yeast have lost their outer wall, the water should lyse the cells (leaving obvious ghosts around), while the SCE doesn't.

Solutions

YPD

  2% dextrose (aka D-glucose, aka glucose)
  2% peptone
  1% yeast extract

SCE ********