Joyce: Preparing RNA from yeast

Concept
The idea is to first grow yeast cells to an appropriate stage to extract RNA from them. There are three stages: the lag, log and stationary phase. During the lag phase, **********

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

Solutions
YPD

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

SCE ********