User:Karmella Haynes/Notebook/Polycomb project/2010/06/09

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06/09/10

  • Growth assay: cell count #2 (start over, pending C12-FDG results, this assay may not be necessary)
  • ✓ Senescence assay optimization: add C12-FDG, microscopy (Silver scope)
  • ✓ ChIP: phenol-chloroform extraction, etOH precipitation (pending Western blot analysis)
    • Discard samples, pull-down unsuccessful



Senescence assay optimization > Use the following C12-FDG concentrations
1-4: 15 μg/mL (20 μL)
5-8: 30 μg/mL (40 μL)
9-12: 60 μg/mL (80 μL)
--> Incubate @ 37°C/ 1 hr.

--> Result: non-specific signal in all samples. C12-FDG not washed off? pH problem?
--> Try flow cytometry of Rotenone stressed vs. non-stressed cells



ChIP
> IP unsuccessful (see Western blot, 6/09/10)
> Reviewed protocol w/ Qingqing and corrected errors
--> Used too little chromatin (use at least 1/4 chromatin prep per IP)
--> Used too few beads for chromatin clearing and IP
--> Non-specific binding wash #5 should be TE + protease inhibitors, not TSE I

> Try again using the 126-1 chromatin prep (test four ab's: IgG, PolII, myc, H3K27me3)