User:Karmella Haynes/Notebook/Polycomb project/2010/05/17

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05/17/10

 * &#x2713; Western: U2OS protein prep from 2x 10 cm plates
 * &#x2713; Cell culture: split U2OS plain 1:20; expand Pc-ATF neg crtl's and set up 6-well plates for Western
 * &#x2713; Growth assay: set up plates

U2OS protein prep > Scrape cells from one plate in 250 μL RIPA buffer (2 plates). Transfer to 1.5 mL tubes. > Rotate at 4°C/ 1 hr. > Spin @ 15,000 rpm/ 4°C/ 15 min. > Transfer sup. to new tube. Combine 2 samples. > Bradford assay:
 * 1) 0 BSA
 * 2) 1 μL 1mg/mL BSA
 * 3) 2 μL "
 * 4) 4 μL "
 * 5) 8 μL "
 * 6) 16 μL "
 * 7) 1 μL U2OS protein prep
 * 8) 2 μL U2OS protein prep



--> Use known amounts of U2OS protein for Western to optimize loading for detecting H3K27me3 and other ab's.

Pc-ATF negative control lines > Choose 3 best growing clones for each line > Use 1/4 for dox-, 1/4 for dox+ in 6-well plates, and 1/2 for 10 cm plate expansion (for frozen stocks)
 * KAH154-1, 2, 4
 * KAH155-2, 5, 6
 * KAH156-2, 3, 5
 * KAH157-1, 2, 5
 * KAH158-2, 3, 4
 * KAH159-1, 2, 3

Growth assay > Harvest cells in 10 mL final vol.; do cell count (Scepter cell counter, use 10x dilution in PBS) > For each line: --> Falcon tube 1: add 25000 cells to 10 mL U2OS plain medium --> Falcon tube 2: add 25000 cells to 10 mL U2OS plain medium; Add 10 μL dox (final conc = 1μg/mL) > Aliquot 2 mL of each to one well in a 12-well plate (5000 cells per well) > For each line, -/+ dox, do cell counts after 2, 4, 6, and 8 days
 * 1) KAH126-1 = count x 10 = 2.007 x105
 * 2) KAH126-3 = 3.137 x105
 * 3) KAH126-4 = 3.302 x105
 * 4) KAH132-8 = 2.584 x105
 * 5) Flp-in T-REx = 2.606 x105


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