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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 4oC.
- 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/37oC
- +5 ul proteinase K stock, non diluted --60min/50oC
- Shoot in 900 ul of 15ug/ml propidium iodide (stock near EtBr, 4oC) in 50 mM Tris pH 7.5 (2.5mg/ml in water [166X])
- sonicate low setting, 3-5 sec. store DARK, 4oC. 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.
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ChIP Protocol-Mechanical