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

From OpenWetWare
Jump to: navigation, search
(Autocreate 2013/07/06 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
 
(mm/dd/yy)
Line 8: Line 8:
  
  
==mm/dd/yy==
+
==07/06/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
* Line item 1
 
* Line item 1
 +
  
 
----
 
----
'''Line item 1'''<br>
+
'''Luciferase activity assay - time point: 4 days'''<br>
> Samples
+
 
 +
 
 +
<u>Cell prep</u>
 +
* 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
 +
 
 +
 
 +
<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])
 +
 
 +
 
 +
<u>Luc assay</u>
 +
* 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 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/07/04 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 || 13,844 || x5 = || 69,220
 
|-
 
|-
| reagent 2 || #
+
| sample 2 || 12,286 || x5 = || 61,430
 
|-
 
|-
| reagent 3 || #
+
| sample 3 || 15,784 || x5 = || 78,920
 
|-
 
|-
| reagent 4 || #
+
| sample 4 || 16,323 || x5 = || 81,615
 
|-
 
|-
| dH<sub>2</sub>O || #
+
| sample 5 || 16,545 || x5 = || 82,725
 
|-
 
|-
| &nbsp; || # μL
+
| sample 6 || 17,885  || x5 = || 89,425
 
|}
 
|}
 
--> Reaction conditions
 
  
  

Revision as of 15:02, 6 July 2013

Owwnotebook icon.pngPc-TF Genomics <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>


07/06/13

  • Line item 1



Luciferase activity assay - time point: 4 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 13,844 x5 = 69,220
sample 2 12,286 x5 = 61,430
sample 3 15,784 x5 = 78,920
sample 4 16,323 x5 = 81,615
sample 5 16,545 x5 = 82,725
sample 6 17,885 x5 = 89,425