User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/04

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07/04/13

  • Gal4-EED/luc time/dose - luc assay & cell counts
  • Happy July 4th! :)
  • Gal4-EED/luc Dox induction #2

Luciferase activity assay - time point: 4 days


Cell prep

  • Harvested cells (induced on 6/30/13)
    • Seeded new 2 new plates with non-induced cells from 6/30/13
    • 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 13,844 x5 = 69,220
sample 2 12,286 x5 = 61,430
sample 3 15,784 x5 = 78,920
sample 4 16,323 x5 = 81,615
sample 5 16,545 x5 = 82,725
sample 6 17,885 x5 = 89,425



Gal4-EED/luc Dox induction #2

  • Lower range worked very well for trail #1! Saw ranges in expression for 1.0 - 10-4 μg/mL doc after 4 days
  • Repeat induction and test after 48 hours
  1. 1.0 μg/mL dox
  2. 10-1
  3. 10-2
  4. 10-3
  5. 10-4
  6. 0

Addition of serial dilutions of dox to cells

  • Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary.
  • Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes...
  tube 1 2 3 4 5 6
1 mg/mL dox (μL) 8.0 --- --- --- --- ---
mixture from prior tube (μL) --- 100 100 100 100 ---
+growth med. (μL) 1000 900 900 900 900 ---
Final [dox] (μg/mL) in 4 mL med. 1.0 10-1 10-2 10-3 10-4 0




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