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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==mm/dd/yy==
==06/19/13==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* 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 22:46, 26 September 2017

Pc-TF Genomics Main project page
Previous entry      Next entry


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)



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