User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/18: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 15: Line 15:
----
----
'''Luciferase activity assay - time point 1'''<br>
'''Luciferase activity assay - time point 1'''<br>
<u>Cell prep</u>
* Harvested cells
* Harvested cells
** Seeded new dox+ plate with 1/2 of cells
** Seeded new dox+ plate with 1/2 of cells
** Pelleted other half, resuspended in 2 mL FACS buffer
** Pelleted other half, resuspended in 2 mL FACS buffer


<u>Assay reagents</u>
* Used the Biotium Steady-Luc Firefly HTS Assay Kit
* Used the Biotium Steady-Luc Firefly HTS Assay Kit
* Thawed reagents to room temp
* 6 experimental samples x 3 repliates = '''18 assays (one time point)'''
* 6 experimental samples x 3 repliates = '''18 assays (one time point)'''
* Need 100 μL complete steady-luc buffer per assay = '''1800 μL complete steady-luc buffer'''
* Need 100 μL complete steady-luc buffer per assay = '''1800 μL complete steady-luc buffer'''
* Dissolve dry D-luciferin to make 50x stock by adding 80 μL dH<sub>2</sub>O to the vial
* Dissolved dry D-luciferin to make 50x stock by adding 80 μL dH<sub>2</sub>O to the vial
* Made 2000 μL complete steady-luc buffer: Add 40 μL 50x D-luciferin to 2 mL buffer
 
<u>Luc assay</u>


* Make 2000 μL complete steady-luc buffer: Add 40 μL 50x D-luciferin to 2 mL buffer
<u>Cell count</u>
* Wang lab's Accuri flow cytometer. Set machine to sample 2 uL of cells




Revision as of 17:54, 18 June 2013

Pc-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>


06/18/13

  • Luciferase activity assay


Luciferase activity assay - time point 1


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
  • Thawed reagents to room temp
  • 6 experimental samples x 3 repliates = 18 assays (one time point)
  • Need 100 μL complete steady-luc buffer per assay = 1800 μL complete steady-luc buffer
  • Dissolved dry D-luciferin to make 50x stock by adding 80 μL dH2O to the vial
  • Made 2000 μL complete steady-luc buffer: Add 40 μL 50x D-luciferin to 2 mL buffer

Luc assay

Cell count

  • Wang lab's Accuri flow cytometer. Set machine to sample 2 uL of cells


Reagent Volume
reagent 1 # μL
reagent 2 #
reagent 3 #
reagent 4 #
dH2O #
  # μL

--> Reaction conditions



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/06/18&oldid=703753"