User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/18

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'''Luciferase activity assay - time point 1'''<br>
'''Luciferase activity assay - time point 1'''<br>
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 +
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<u>Cell prep</u>
* Harvested cells
* Harvested cells
** Seeded new dox+ plate with 1/2 of cells
** Seeded new dox+ plate with 1/2 of cells
** Pelleted other half, resuspended in 2 mL FACS buffer
** Pelleted other half, resuspended in 2 mL FACS buffer
 +
 +
<u>Assay reagents</u>
* Used the Biotium Steady-Luc Firefly HTS Assay Kit
* Used the Biotium Steady-Luc Firefly HTS Assay Kit
 +
* Thawed reagents to room temp
* 6 experimental samples x 3 repliates = '''18 assays (one time point)'''
* 6 experimental samples x 3 repliates = '''18 assays (one time point)'''
* Need 100 μL complete steady-luc buffer per assay = '''1800 μL complete steady-luc buffer'''
* Need 100 μL complete steady-luc buffer per assay = '''1800 μL complete steady-luc buffer'''
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* Dissolve dry D-luciferin to make 50x stock by adding 80 μL dH<sub>2</sub>O to the vial
+
* Dissolved dry D-luciferin to make 50x stock by adding 80 μL dH<sub>2</sub>O to the vial
 +
* Made 2000 μL complete steady-luc buffer: Add 40 μL 50x D-luciferin to 2 mL buffer
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 +
<u>Luc assay</u>
-
* Make 2000 μL complete steady-luc buffer: Add 40 μL 50x D-luciferin to 2 mL buffer
+
<u>Cell count</u>
 +
* Wang lab's Accuri flow cytometer. Set machine to sample 2 uL of cells

Revision as of 19:54, 18 June 2013

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06/18/13

  • Luciferase activity assay



Luciferase activity assay - time point 1


Cell prep

  • Harvested cells
    • Seeded new dox+ plate with 1/2 of cells
    • Pelleted other half, resuspended in 2 mL FACS buffer


Assay reagents

  • Used the Biotium Steady-Luc Firefly HTS Assay Kit
  • Thawed reagents to room temp
  • 6 experimental samples x 3 repliates = 18 assays (one time point)
  • Need 100 μL complete steady-luc buffer per assay = 1800 μL complete steady-luc buffer
  • Dissolved dry D-luciferin to make 50x stock by adding 80 μL dH2O to the vial
  • Made 2000 μL complete steady-luc buffer: Add 40 μL 50x D-luciferin to 2 mL buffer

Luc assay

Cell count

  • Wang lab's Accuri flow cytometer. Set machine to sample 2 uL of cells


Reagent Volume
reagent 1 # μL
reagent 2 #
reagent 3 #
reagent 4 #
dH2O #
  # μL

--> Reaction conditions



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