Untitled Document

FACS (Bob)

Cells (0.3-0.4 OD₆₀₀); 1mL. Spin down, resuspend in 300ul 50 mM Tris pH 7.5 and add 700 ul EtOH. Keeps > 1week at 4^oC.
Spin out EtOH, aspirate "dry" (wash with 50 mM Tris pH 7.5 once to ensure all EtOH gone)
Resuspend in 100 ul RNAse (10mg/mL in 50 mM Tris pH 7.5) ---2H/37^oC
+5 ul proteinase K stock, non diluted --60min/50^oC
Shoot in 900 ul of 15ug/ml propidium iodide (stock near EtBr, 4^oC) in 50 mM Tris pH 7.5 (2.5mg/ml in water [166X])
sonicate low setting, 3-5 sec. store DARK, 4^oC. Can be used up to 6 months

Koshland Web Site/Methods
FLOW CYTOMETRY
FACS (Bob) Cells (0.3-0.4 OD₆₀₀); 1mL. Spin down, resuspend in 300ul 50 mM Tris pH 7.5 and add 700 ul EtOH. Keeps > 1week at 4^oC. Spin out EtOH, aspirate "dry" (wash with 50 mM Tris pH 7.5 once to ensure all EtOH gone) Resuspend in 100 ul RNAse (10mg/mL in 50 mM Tris pH 7.5) ---2H/37^oC +5 ul proteinase K stock, non diluted --60min/50^oC Shoot in 900 ul of 15ug/ml propidium iodide (stock near EtBr, 4^oC) in 50 mM Tris pH 7.5 (2.5mg/ml in water [166X]) sonicate low setting, 3-5 sec. store DARK, 4^oC. Can be used up to 6 months
Comments: H₂O can be substituted for Tris in steps 1,2. Bob uses 400 µl of Propidium Iodide.
Koshland Lab Home Page \| Berkeley Faculty Page