This is a cached page for the URL (http://cancer.ucsd.edu/flow/Protocols/Yeast%20Cell%20Cycle%20SYBR.asp). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
SYBR Green I Yeast Cell Cycle

Flow cytometry main page

Protocols main page

<---Back to Flow Cyto Home

<--- Back to Protocols

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
50 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 50 C.
  5. Add 20 microliters Proteinase K solution and incubate 1 hour at 50 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.

 <---Back to Flow Cyto Home

<--- Back to Protocols

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

 


Home  |  Events  |  Friends & Supporters  |  Faculty & Staff site |  Search

This site is a service of the Moores UCSD Cancer Center.  Comments or questions?  Please contact our webmaster.
Help using this site.