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Yeast Cell Cycle with SYBR Green I

SYBR Green I is better than PI.

Materials

RNAse

2 mg/ml RNAse A in 50 mM Tris pH 8.0 & 15 mM NaCl
(boil for 5 -10 minutes and allow to cool, aliquot and freeze)

Proteinase K

20 mg/ml in H2O

Tris-EDTA

3.7 g Tris base
121 mg EDTA
in 1 liter H2O pH 8.0

SYBR Green I Staining Solution

1:10 dilution of SYBR Green I in Tris-EDTA with 0.25% (v/v) Triton X-100

Sodium Citrate Buffer

14.7 g sodium citrate in 1 liter H2O pH 7.5

50º C waterbath

Method

  1. Pellet 107 cells and re-suspend in 1.5 ml H2O.
  2. Slowly add 3.5 ml 100% EtOH. Fix at least 1 hr at Room Temperature (or overnight in the refrigerator).
  3. Wash fixed cells once with sodium citrate buffer and transfer to Falcon® 12 X 75 polystyrene tubes [35]2052, 2054, or 2058.(DO NOT SUBSTITUTE TUBES.)
  4. Re-suspend in 0.5 ml RNAse solution and incubate for 1 - 2 hours at 50º C.
  5. Add 20 microliters Proteinase K solution and incubate 1 hour at 50º C.
  6. Add 20 - 100 microliters of SYBR Green I Staining Solution and incubate overnight in the refrigerator.
  7. Sonicate and analyze.

Reference

Fortuna M, Joao Sousa M, Corte-Real M, and Leao C. in Current Protocols in Cytometry, UNIT 11.13.