YEAST CELL CYCLE WITH SYTOX GREEN
- Grow cells under the desired conditions to 0.5-1x107 cells/mL. Collect up to 1-2x107 cells per sample. All the following steps can be performed in a single 1.7mL microfuge tube per sample.
- Pellet the cells (a few seconds in a microfuge). Resuspend the cells in 300μl H2O. Be sure the pellets have been dispersed before the next step.
- Add 0.7mL 100% ethanol and vortex; incubate at 4°C for at least 30 minutes.
- Pellet the cells as before and resuspend in 1mL of 50mM sodium citrate, pH 7.4.
- Sonicate the cells (10 pulses of 0.9 seconds each, setting of –32 to –40).
- Pellet the cells again and resuspend them in 1mL Na-citrate containing 0.25mg/mL RNase A. Incubate at 50-55°C for 1 hour.
- Add 25μL of 20mg/mL Proteinase K and continue the incubation for an additional hour.
- Pellet the cells again and resuspend the pellets in 500μl of 50mM Na-citrate. [But go instead to “Alternative” in Step 10 if your cell concentration is not very high.]
- Prepare a batch of 1x Sytox Green (= 1μM) in 50mM Na-citrate. The stock solution provided by the vendor is 5mM = 5000x, in DMSO. Note: the manufacturer recommends wearing two pairs of gloves when handling the stock solution. The Sytox stock solution takes a while to thaw, so plan ahead.
- Pipette out 1 ml aliquots of the 1x Sytox solution into FACS analysis tubes (Falcon 2054). Add 50μL of the cell sample (or enough to get ~1-2x106cells; the goal is to have 100-200 events per second within the gated region when the samples are being read). Incubate 1 hour to overnight at 4°C; stained samples can be stored at 4°C for several weeks. [Alternative: If your sample is low-concentration, you can simply add 1 ml of 2x Sytox to the cell sample immediately after step 7, and you’ll be good to go.]
- Use FL1 on the FACSCAN instrument. Sytox Green fluoresces more strongly than propidium iodide, so the voltage/gain won’t have to be set as high as with propidium iodide.
Sytox Green: Invitrogen, catalog # S7020
RNase A Stock Solution (10mg/mL)
- 0.2g Ribonuclease A (Sigma #R-5503)
- 200µL 1M Tris-HCl, pH 7.5
- 60µL 5M NaCl
- Bring volume up to 20mL with H2O
- Boil 15 minutes.
- Let cool slowly to room temperature.
- Aliquot and store at -20°C.
Proteinase K Stock Solution (20mg/mL)
- Stock solution is 20mg/mL in
- 10mM Tris pH 8.0
- 1mM CaCl2
- 50% glycerol
- To make 5mL of stock solution, dissolve 100mg Proteinase K in:
- 3.125mL 80% glycerol*
- 50μl 1 M Tris pH 8.0
- 50μl 100mM CaCl2
- 1.775mL H2O
- = 5mL total; dispense in 1mL portions and store at –20°C.
- *autoclaved stock; easier to deal with than 100% glycerol that’s not been sterilized