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

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06/29/10

  • ✓ ChIP: cross-link 126-1, 132-8 (confirmed positive RFP expression via microscopy)
  • ✓ ChIP: myc-bead pull-down
  • ✓ 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)
  • ✓ Culture: split 129-4, 131-9, 126-1, 126-3, 132-8, 154-2, FTRx 1:10
  • ✓ Western: 129-4, 131-9 -/+ dox 6-well plate
  • ✓ 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:

  1. 500 μL chromatin + 10 μL myc-bead slurry
  2. 500 μL chromatin + 10 μL sepharose slurry

--> Rotate at 4°C overnight



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