Yeast Cell Cycle with Sytox Green

 

1. Grow cells under the desired conditions to 0.5-1 x 107 cells/ml. Collect up to 1-2 x 107 cells per sample. All the following steps can be performed in a single 1.7 ml microfuge tube per sample.

2. Pellet the cells (a few seconds in a microfuge). Resuspend the cells in 300 μl H2O. Be sure the pellets have been dispersed before the next step.

3. Add 0.7 ml 100% ethanol and vortex; incubate at 4°C for at least 30 minutes.

4. Pellet the cells as before and resuspend in 1 ml of 50 mM sodium citrate, pH 7.4.

5. Sonicate the cells (10 pulses of 0.9 seconds each, setting of –32 to –40).

6. Pellet the cells again and resuspend them in 1 ml Na-citrate containing 0.25 mg/ml RNase A. Incubate at 50-55°C for 1 hour.

7. Add 25 μl of 20 mg/ml Proteinase K and continue the incubation for an additional hour.

8. Pellet the cells again and resuspend the pellets in 500 μl of 50 mM Na-citrate. [But go instead to “Alternative” in Step 10 if your cell concentration is not very high.]

9. Prepare a batch of 1x Sytox Green (= 1 μM) in 50 mM Na-citrate. The stock solution provided by the vendor is 5 mM = 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.

10. Pipette out 1 ml aliquots of the 1x Sytox solution into FACS analysis tubes (Falcon 2054). Add 50 μl of the cell sample (or enough to get ~1-2 x 106 cells; 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.]

11. 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 (10 mg/ml)

0.2 g Ribonuclease A (Sigma #R-5503)

200 uL 1 M Tris-HCl, pH 7.5

60 uL 5M NaCl

Bring volume up to 20 ml with H2O

Boil 15 minutes.

Let cool slowly to room temperature.

Aliquot and store at -20°C.

 

Proteinase K stock solution (20 mg/ml)

Stock solution is 20 mg/ml in

10 mM Tris pH 8.0

1 mM CaCl2

50% glycerol

To make 5 ml of stock solution, dissolve 100 mg Proteinase K in:

3.125 ml 80% glycerol*

50 μl 1 M Tris pH 8.0

50 μl 100 mM CaCl2

1.775 ml H2O

= 5 ml total; dispense in 1 ml portions and store at –20°C.

*autoclaved stock; easier to deal with than 100% glycerol that’s not been sterilized