User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/02/22: Difference between revisions
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|style="background-color: #F2F2F2" align="center"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==02/23/13== | ||
* | * DAPI/ luciferase staining | ||
* New cell plates | |||
---- | |||
'''DAPI/ luciferase staining'''<br> | |||
* D-luciferin stock solution: 30mg/ml in 1xPBS, working solution is 150ug/ml (200x) | |||
* Hoescht: made by Dr. Haynes; 5000x in sterile dH<sub>2</sub>O | |||
* Diluted Hoescht and D-luc to working concentration (1x) in 1xPBS. Added 2 mL to each well... | |||
Used 3 wells: | |||
# DAPI + D-luc | |||
# DAPI only | |||
# PBS | |||
* Let cells sit at room temperature for 10 minutes in the dark to develop signal | |||
Results/ Conclusions: | |||
* 2 mL medium is susceptible to drying at outer wells; disrupts cell growth | |||
* Did not detect D-luc signal. Perhaps D-luc imaging should start immediately after adding reagents. The D-luc signal has a short half-life. | |||
* Cells rounded up a bit after sitting in PBS. Next time use 10% FBS in PBS, or try DMEM without red dye. | |||
* DAPI signal was barely visible | |||
Note: add photos here | |||
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'''New cell plates'''<br> | |||
Dr. Haynes; For more practice staining (Monday) | |||
* HEK293 luc #4, 6-well plate, ~1x10<sup>5</sup> cells/ well, 4 mL growth medium | |||
* HEK293 Gal4-EED, 6-well plate, ~1x10<sup>5</sup> cells/ well, 4 mL growth medium | |||
Stock cultures (T75 flasks) | |||
* HEK293 luc #4, 1:5 | |||
* HEK293 Gal4-EED, 1:4 | |||
* HEK293 Gal4-EED, 1:4 in 0.5 μg/mL puromycin (to select for Gal4-EED RNAi tuner) | |||
Latest revision as of 22:29, 26 September 2017
Project name | Main project page Previous entry Next entry |
02/23/13
DAPI/ luciferase staining
Used 3 wells:
Results/ Conclusions:
New cell plates
Stock cultures (T75 flasks)
|