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

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05/17/10

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

File:KAH 051710 chart1.tif

--> 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

  • KAH154-1, 2, 4
  • KAH155-2, 5, 6
  • KAH156-2, 3, 5
  • KAH157-1, 2, 5
  • KAH158-2, 3, 4
  • KAH159-1, 2, 3

> Use 1/4 for dox-, 1/4 for dox+ in 6-well plates, and 1/2 for 10 cm plate expansion (for frozen stocks)



Growth assay
> Harvest cells in 10 mL final vol.; do cell count (Scepter cell counter, use 10x dilution in PBS)

  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

> 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