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

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(Autocreate 2013/07/06 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
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==mm/dd/yy==
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==07/06/13==
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'''Line item 1'''<br>
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'''Luciferase activity assay - time point: 4 days'''<br>
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> Samples
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<u>Cell prep</u>
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* Harvested cells (induced on 7/04/13)
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** Seeded new 2 new plates with non-induced cells from 7/04/13
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** Pelleted all of the dox+ cells, resuspended in 2 mL FACS buffer
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<u>Assay reagents</u>
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* 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])
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<u>Luc assay</u>
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* Filtered '''700 μL cells''' through strainer caps
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* Used opaque white Costar plate (from Rege lab)
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* Samples loaded in triplicate (by columns)
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* Included luc buffer + FACS-buffer "blank" sample (well D1)
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* Other steps same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/07/04 6/18/13]
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<u>Cell counts</u>
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* Wang lab's Accuri flow cytometer.
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* Set machine to read 20 uL of cells
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* Be sure to "clean" with water-run in between samples
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{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
|-valign="top"
|-valign="top"
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| <u>Reagent</u> || <u>Volume</u>  
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| <u>Sample ID</u> || <u>Gated count/ 20 μL</u> || &nbsp; || <u>Cells/ 100 μL</u>
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|-
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| reagent 1 || # μL
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| sample 1 || 13,844 || x5 = || 69,220
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|-
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| reagent 2 || #
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| sample 2 || 12,286 || x5 = || 61,430
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|-
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| reagent 3 || #
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| sample 3 || 15,784 || x5 = || 78,920
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|-
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| reagent 4 || #
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| sample 4 || 16,323 || x5 = || 81,615
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|-
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| dH<sub>2</sub>O || #
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| sample 5 || 16,545 || x5 = || 82,725
|-
|-
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| &nbsp; || # μL
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| sample 6 || 17,885  || x5 = || 89,425
|}
|}
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--> Reaction conditions
 

Revision as of 17:02, 6 July 2013

Pc-TF Genomics Main project page
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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



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