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

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(07/06/13)
Current revision (06:33, 9 July 2013) (view source)
(07/06/13)
 
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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
+
* SK-N-SH +PcTF & mock - TRIzol prep #1
Line 46: Line 46:
| <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 || 37,629 || x5 = ||  
+
| sample 1 || 37,629 || x5 = || 188,145
|-
|-
-
| sample 2 || 34,580 || x5 = || ###
+
| sample 2 || 34,580 || x5 = || 172,900
|-
|-
-
| sample 3 || 40,096 || x5 = ||  
+
| sample 3 || 40,096 || x5 = || 200,480
|-
|-
-
| sample 4 || 46,599 || x5 = ||  
+
| sample 4 || 46,599 || x5 = || 232,995
|-
|-
-
| sample 5 || 46,491 || x5 = ||  
+
| sample 5 || 46,491 || x5 = || 232,455
|-
|-
-
| sample 6 ||   || x5 = ||  
+
| sample 6 || 46,254  || x5 = || 231,270
|}
|}
 +
 +
 +
----
 +
 +
'''SK-N-SH TRIzol prep'''<br>
 +
# +PcTF, plate 1
 +
# mock, plate 4
 +
(Cells may need longer to express genes. Previous expt. was done after ~10 days. Will do this prep just in case cell culture declines)
 +
 +
* Used other two plates to passage cells ~1:5 (will prep after returning from SB6.0)
 +
 +
* Cells were 100% confluent
 +
* Discarded growth medium
 +
* Added 2 mL TRIzol directly to plates
 +
* Incubated at r.t. for ~5 min.
 +
* Collected lysed cells from plate with gentle scraping (pipette tip)
 +
* Transferred 500 μL aliquots to 2.0 mL tubes. Stored at -80°C
 +
** 4x PcTF+ samples
 +
** 4x mock samples
 +
 +

Current revision

Pc-TF Genomics Main project page
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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 & mock - 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 = 188,145
sample 2 34,580 x5 = 172,900
sample 3 40,096 x5 = 200,480
sample 4 46,599 x5 = 232,995
sample 5 46,491 x5 = 232,455
sample 6 46,254 x5 = 231,270



SK-N-SH TRIzol prep

  1. +PcTF, plate 1
  2. mock, plate 4

(Cells may need longer to express genes. Previous expt. was done after ~10 days. Will do this prep just in case cell culture declines)

  • Used other two plates to passage cells ~1:5 (will prep after returning from SB6.0)
  • Cells were 100% confluent
  • Discarded growth medium
  • Added 2 mL TRIzol directly to plates
  • Incubated at r.t. for ~5 min.
  • Collected lysed cells from plate with gentle scraping (pipette tip)
  • Transferred 500 μL aliquots to 2.0 mL tubes. Stored at -80°C
    • 4x PcTF+ samples
    • 4x mock samples




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