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

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(06/18/13)
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* 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'''
* Dissolved 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
+
 
<u>Luc assay</u>
<u>Luc assay</u>
 +
* Filtered 500 μL cells through strainer caps (<font color="red">should use 700 μL next time</font>)
 +
* Transferred 100 μL strained cells to 3 wells in 96-well plate (black walls; clear, flat bottom)
 +
** Saved remainder of strained cells for flow cytometry/ counting
 +
* Made 2000 μL complete steady-luc buffer: Added 40 μL 50x D-luciferin to 2 mL buffer
 +
* QUICKLY Added 100 μL of complete steady-luc buffer to each sample (pipetted up/down once to mix)
 +
* Incubated at room temp/ 10 min.
 +
<u>Cell count</u>
<u>Cell count</u>

Revision as of 20:35, 18 June 2013

Pc-TF Genomics Main project page
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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


Luc assay

  • Filtered 500 μL cells through strainer caps (should use 700 μL next time)
  • Transferred 100 μL strained cells to 3 wells in 96-well plate (black walls; clear, flat bottom)
    • Saved remainder of strained cells for flow cytometry/ counting
  • Made 2000 μL complete steady-luc buffer: Added 40 μL 50x D-luciferin to 2 mL buffer
  • QUICKLY Added 100 μL of complete steady-luc buffer to each sample (pipetted up/down once to mix)
  • Incubated at room temp/ 10 min.


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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