User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/03/01: Difference between revisions
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* | HEK293 Luc #4 DAPI staining | ||
Assay on 96-well plate | |||
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* 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 | |||
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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 | |||
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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 | |||
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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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03/01/13HEK293 Luc #4 DAPI staining Assay on 96-well plate
Results:
Literature review results:
Next steps:
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