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Yeast Cell Cycle
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
- Pellet 107
cells and re-suspend in 1.5 ml H2O.
- Slowly add 3.5 ml 100%
EtOH. Fix at least 1 hr at Room Temperature (or overnight in the refrigerator).
- 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.)
- Re-suspend in 0.5 ml RNAse
solution and incubate for 1 - 2 hours at 50º C.
- Add 20 microliters Proteinase
K solution and incubate 1 hour at 50º C.
- Add 20 - 100 microliters
of SYBR Green I Staining Solution and incubate overnight in the refrigerator.
- Sonicate and analyze.
Reference
Fortuna
M, Joao Sousa M, Corte-Real M, and Leao C. in Current Protocols in Cytometry,
UNIT 11.13.
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Dennis J. Young,
UCSD Cancer Center Flow Cytometry Core Facility
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