User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/06: Difference between revisions

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




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<u>Assay reagents</u>
<u>Assay reagents</u>
* Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/07/04 6/18/13])
* Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/06/18 6/18/13])




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* Samples loaded in triplicate (by columns)
* Samples loaded in triplicate (by columns)
* Included luc buffer + FACS-buffer "blank" sample (well D1)
* Included luc buffer + FACS-buffer "blank" sample (well D1)
* Other steps same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/07/04 6/18/13]
* Other steps same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/06/18 6/18/13]




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| <u>Sample ID</u> || <u>Gated count/ 20 μL</u> || &nbsp; || <u>Cells/ 100 μL</u>
| <u>Sample ID</u> || <u>Gated count/ 20 μL</u> || &nbsp; || <u>Cells/ 100 μL</u>
|-
|-
| sample 1 || ### || x5 = || ###
| sample 1 || 37,629 || x5 = ||  
|-
|-
| sample 2 || ### || x5 = || ###
| sample 2 || 34,580 || x5 = || ###
|-
|-
| sample 3 || 15,784 || x5 = || 78,920
| sample 3 || 40,096 || x5 = ||  
|-
|-
| sample 4 || 16,323 || x5 = || 81,615
| sample 4 || 46,599 || x5 = ||  
|-
|-
| sample 5 || 16,545 || x5 = || 82,725
| sample 5 || 46,491 || x5 = ||  
|-
|-
| sample 6 || 17,885 || x5 = || 89,425
| sample 6 || 46,254 || x5 = ||  
|}
|}


Revision as of 20:48, 6 July 2013

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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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