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)
 * &#x2713; Senescence assay optimization: add C12-FDG, microscopy (Silver scope)
 * &#x2713; 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)


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