User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/03/01: Difference between revisions

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==Entry title==
==03/01/13==
* Insert content here...
HEK293 Luc #4 DAPI staining
Assay on 96-well plate
 
 
 
----
* DAPI/Hoescht: made by Dr. Haynes; 5000x in sterile dH<sub>2</sub>O
* Diluted Hoescht to working concentration (1x) in 1xPBS.  
* Added 2 mL to each well
* Let sit at room temperature for 5, 10, 20, and 30 mins in the dark to develop signal
 
 
----
Results:
* strong fluorescence signal only in dead/detached cells
* slightly better signal after 5 mins, no increase in signal afterwards
* still too weak signal in living cells
 
* 96-well plate resulted in few distinct good colonies
** problematic when too close to the wall
** takes longer time to grow
** often wells turned out empty or cells died
** but a few very good nicely formed colonies
 
 
----
Literature review results:
* DAPI doesn't stain replicating DNA, yet it's one of the best nuclei staining dyes.
* luciferin signal is only detectable with very sensitive liquid gas cooled cameras
 
----
Next steps:
* Detect luciferin signal with antibody staining instead of direct bioluminescence
* Therefore fixing cells with formaldehyde
* Should improve DAPI signal




Revision as of 21:43, 18 March 2013

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03/01/13

HEK293 Luc #4 DAPI staining Assay on 96-well plate


  • DAPI/Hoescht: made by Dr. Haynes; 5000x in sterile dH2O
  • Diluted Hoescht to working concentration (1x) in 1xPBS.
  • Added 2 mL to each well
  • Let sit at room temperature for 5, 10, 20, and 30 mins in the dark to develop signal


Results:

  • strong fluorescence signal only in dead/detached cells
  • slightly better signal after 5 mins, no increase in signal afterwards
  • still too weak signal in living cells
  • 96-well plate resulted in few distinct good colonies
    • problematic when too close to the wall
    • takes longer time to grow
    • often wells turned out empty or cells died
    • but a few very good nicely formed colonies


Literature review results:

  • DAPI doesn't stain replicating DNA, yet it's one of the best nuclei staining dyes.
  • luciferin signal is only detectable with very sensitive liquid gas cooled cameras

Next steps:

  • Detect luciferin signal with antibody staining instead of direct bioluminescence
  • Therefore fixing cells with formaldehyde
  • Should improve DAPI signal



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