User:Karmella Haynes/Notebook/Polycomb project/2010/06/07

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06/07/10

  • ✓ Growth assay: Cell count #1 (Scepter hand-held counter) harvest cells in 1 mL medium total, count 1:4 dilution in 1x PBS
  • ✓ Pc-ATF + H3met sensor lines: Use back-up 6-well transfectants to make 500k cell plates (very little growth in 100k cell plates); make back-up 6-well w/ 1/4 of cells (U2OS plain medium)
  • ✓ ChIP trial: block beads, pre-clear chromatin (U2OS plain), antibody binding (mouse PolII, mouse IgG)



Growth assay
> Checked cells for RFP signal
--> uninduced Pc-ATF lines showed a few RFP+ cells (< 10%)
--> Nearly 100% of cells show RFP+ with dox treatment
> Accidentally resuspended sample #8 (132-8 +dox) in ~1.5 mL medium (adjust cell count accordingly)



ChIP trial
> Follow protocol from Qingqing
> Use U2OS plain sample
> Antibodies

  1. Mouse Pol II (ab817, 1.0 μg/μL): 5 μL (5 μg)
  2. Mouse IgG neg. crtl. (sc-2025, 0.4 μg/μL): 10 μL (4 μg)


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