Difference between revisions of "User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/19"

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(Autocreate 2013/06/19 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
 
(06/19/13)
 
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==mm/dd/yy==
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==06/19/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
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* Luc assay, time point 2
 +
* PcTF transfection for U2OS, outgrowth #2
 +
 
  
 
----
 
----
'''Line item 1'''<br>
+
'''Luciferase activity assay - time point 2'''<br>
> Samples
+
 
 +
 
 +
<u>Cell prep</u>
 +
* Harvested cells
 +
** Seeded new dox+ plate with 1/2 of cells
 +
** Pelleted other half, resuspended in 2 mL FACS buffer
 +
 
 +
 
 +
<u>Assay reagents</u>
 +
* Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as 6/18/13)
 +
 
 +
 
 +
<u>Luc assay</u>
 +
* Filtered '''700 μL cells''' through strainer caps
 +
* Other steps same as 6/18/13
 +
 
 +
 
 +
<u>Cell counts</u>
 +
* Wang lab's Accuri flow cytometer.
 +
* Set machine to read 20 uL of cells
 +
* Be sure to "clean" with water-run in between samples
 +
 
  
 
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
 
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
 
|-valign="top"
 
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>  
+
| <u>Sample ID</u> || <u>Gated count/ 20 μL</u> || &nbsp; || <u>Cells/ 100 μL</u>
 
|-
 
|-
| reagent 1 || # μL
+
| sample 1 || 10,880 || x5 = || 54,400
 
|-
 
|-
| reagent 2 || #
+
| sample 2 || 10,430 || x5 = || 52,150
 
|-
 
|-
| reagent 3 || #
+
| sample 3 || 11,897 || x5 = || 59,485
 
|-
 
|-
| reagent 4 || #
+
| sample 4 || 10,316 || x5 = || 51,580
 
|-
 
|-
| dH<sub>2</sub>O || #
+
| sample 5 || 9,784 || x5 = || 48,920
 
|-
 
|-
| &nbsp; || # μL
+
| sample 6 || 11,549 || x5 = || 57,745
 
|}
 
|}
  
--> Reaction conditions
 
  
 +
 +
----
 +
'''PcTF transfection for U2OS, outgrowth #2'''
 +
 +
* Expanded transfected and mock-transfected cells from old 10 cm plates (6/15/13)
 +
* Used only samples 1 (+PcTF) and 4 (mock)
 +
* 1:10 passage into three new 10 cm plates labeled "1-A, 1-B, 1-C" (+PcTF) and "4-A, 4-B, 4-C" (mock)
  
  

Latest revision as of 20:13, 26 June 2013

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06/19/13

  • Luc assay, time point 2
  • PcTF transfection for U2OS, outgrowth #2



Luciferase activity assay - time point 2


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 (same as 6/18/13)


Luc assay

  • Filtered 700 μL cells through strainer caps
  • 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 10,880 x5 = 54,400
sample 2 10,430 x5 = 52,150
sample 3 11,897 x5 = 59,485
sample 4 10,316 x5 = 51,580
sample 5 9,784 x5 = 48,920
sample 6 11,549 x5 = 57,745



PcTF transfection for U2OS, outgrowth #2

  • Expanded transfected and mock-transfected cells from old 10 cm plates (6/15/13)
  • Used only samples 1 (+PcTF) and 4 (mock)
  • 1:10 passage into three new 10 cm plates labeled "1-A, 1-B, 1-C" (+PcTF) and "4-A, 4-B, 4-C" (mock)