User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/06

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07/06/13

  • Gal4-EED/luc dox time point (48 hours)
  • Gal4-EED/luc PcTF transfection - microscopy & flow cytometry
  • SK-N-SH +PcTF - TRIzol prep #1



Luciferase activity assay - time point: 2 days

Cell prep

  • Harvested cells (induced on 7/04/13)
    • Seeded new 2 new plates with non-induced cells from 7/04/13
    • Pelleted all of the dox+ cells, resuspended in 2 mL FACS buffer


Assay reagents

  • Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as 6/18/13)


Luc assay

  • Filtered 700 μL cells through strainer caps
  • Used opaque white Costar plate (from Rege lab)
  • Samples loaded in triplicate (by columns)
  • Included luc buffer + FACS-buffer "blank" sample (well D1)
  • Other steps same as 6/18/13


Cell counts

  • Wang lab's Accuri flow cytometer.
  • Set machine to read 20 uL of cells
  • Be sure to "clean" with water-run in between samples


Sample ID Gated count/ 20 μL   Cells/ 100 μL
sample 1 37,629 x5 =
sample 2 34,580 x5 = ###
sample 3 40,096 x5 =
sample 4 46,599 x5 =
sample 5 46,491 x5 =
sample 6 46,254 x5 =



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