User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/02/22: Difference between revisions
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* | * DAPI/ luciferase staining | ||
* New cell plates | |||
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'''DAPI/ luciferase staining'''<br> | |||
Note: David, enter formulas, procedures here | |||
* D-luciferin stock solution: ??? in 1xPBS | |||
* 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. | |||
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) | |||
Revision as of 15:39, 23 February 2013
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02/23/13
DAPI/ luciferase staining
Used 3 wells:
Results/ Conclusions:
New cell plates
Stock cultures (T75 flasks)
|