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Yeast Cell Cycle

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Yeast Cell Cycle

Sytox Green 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)
Protease
5 mg/ml pepsin, 4.5 microliter/ml concentrated HCl in H2O
SYTOX Green Staining Solution
1 mM SYTOX Green in 50 mM Tris pH 7.5
 

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 RT (or overnight in the refrigerator).
  3. Wash fixed cells once with 1.0 ml H2O.
  4. Re-suspend in 0.5 ml RNAse solution and incubate for 2 - 12 hours at 37 C.
  5. Pellet and re-suspend in protease solution and incubate 20 minutes at 37 C.
  6. Pellet and re-suspend in 0.5 ml 50 mM Tris pH 7.5.
  7. Can be stored at 4 C.
  8. Add 50 - 100 microliters of processed cells to 500 - 900 microliters of SYTOX Green Staining Solution in Falcon® 12 X 75 polystyrene tubes [35]2052, 2054, or 2058.(DO NOT SUBSTITUTE TUBES.)
  9. Sonicate and analyze.

  Reference
Haase SB, Reed SI. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle. 2002 Mar-Apr;1(2):132-6. PMID: 12429922

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Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility

 


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