User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/15

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(06/15/13)
Current revision (23:11, 26 June 2013) (view source)
(06/15/13)
 
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'''* PcTF transfection for U2OS, outgrowth #1'''
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'''PcTF transfection for U2OS, outgrowth #1'''
* 6-well plates: 1-3 = +PcTF, 4-6 = mock (lipo only)
* 6-well plates: 1-3 = +PcTF, 4-6 = mock (lipo only)
* Expand cells from 6-well plates in individual 10 cm plates (10 mL medium). 6 10 cm plates total.
* Expand cells from 6-well plates in individual 10 cm plates (10 mL medium). 6 10 cm plates total.

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06/15/13

  • Gal4-EED/luc Luciferase ICC - fixation, permeabilization, primary ab staining
  • Gal4-EED/ luc dox induction - shipment 2 cells
  • PcTF transfection for U2OS, outgrowth #1



Gal4-EED/luc Luciferase ICC

Samples (Shipment 1 cells)

  1. 0 μg/mL dox
  2. 0.1
  3. 0.2
  4. 0.5
  5. 1.0
  6. 2.0 (induced on 6/02/13, 13 days)


  • Followed standard protocol
  • Harvested cells from one 6-well plate (split plate #2 for further analyses)
  • At the primary antibody staining step, keep samples and reagents on ice overnight in the cold room



Gal4-EED/luc dox induction

Samples (Shipment 2 cells); new 6-well plate...

  1. 0 μg/mL dox
  2. 0.1
  3. 0.2
  4. 0.5
  5. 1.0
  6. 2.0 (induced on 6/02/13, 13 days)
  • For this set, test earlier time points and try luc activity assay



PcTF transfection for U2OS, outgrowth #1

  • 6-well plates: 1-3 = +PcTF, 4-6 = mock (lipo only)
  • Expand cells from 6-well plates in individual 10 cm plates (10 mL medium). 6 10 cm plates total.
  • Use standard growth medium.
  • Use all cells. Do not dilute.



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