User:Karmella Haynes/Notebook/Polycomb project/2010/06/29

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06/29/10

 * &#x2713; ChIP: cross-link 126-1, 132-8 (confirmed positive RFP expression via microscopy)
 * &#x2713; ChIP: myc-bead pull-down
 * &#x2713; Transfection (H3me reporter lines, trial 2): Plate entire sample in 10 cm plate, 100 μg/mL hygromycin; 4 transfections total; include non-transfected sample as neg. control (5 plates total)
 * &#x2713; Culture: split 129-4, 131-9, 126-1, 126-3, 132-8, 154-2, FTRx 1:10
 * &#x2713; Western: 129-4, 131-9 -/+ dox 6-well plate
 * &#x2713; Senescence assay: FTRx, 2 6-well plates

ChIP: myc-bead pull-down > Try conjugated myc-beads from Cell Signaling (#3400) --> Prepare chromatin (132-8 sonicated 6 mL sample) according to Qingqing's protocol (add PLAAC, PMSF, detergent, salt, etc.) --> Prepare control sepharose beads: pellet 100 μL protein A sepharose (Amersham #17-5280-01); wash with 1xPBS three times; resuspend in 100 μL 1xPBS --> Binding: --> Rotate at 4°C overnight
 * 1) 500 μL chromatin + 10 μL myc-bead slurry
 * 2) 500 μL chromatin + 10 μL sepharose slurry

Senescence assay > For titration of C12-FDG > Seed ~200k cells per well in 6-well plates; ~3.5 mL/ well total volume > Rotenone treatment (~3 days): --> Plate 1: 0 rotenone, 5.0 μL DMSO --> Plate 2: 0.2 μg/mL rotenone (3.5 μL), 1.5 μL DMSO


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