User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/02/22

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(Autocreate 2013/02/22 Entry for User:David_Dreher/Notebook/Chromatin_controlled_cell_pattern)
Current revision (23:53, 18 March 2013) (view source)
(02/23/13)
 
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==Entry title==
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==02/23/13==
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* Insert content here...
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* DAPI/ luciferase staining
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* New cell plates
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----
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'''DAPI/ luciferase staining'''<br>
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* D-luciferin stock solution: 30mg/ml in 1xPBS, working solution is 150ug/ml (200x)
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* Hoescht: made by Dr. Haynes; 5000x in sterile dH<sub>2</sub>O
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* Diluted Hoescht and D-luc to working concentration (1x) in 1xPBS. Added 2 mL to each well...
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Used 3 wells:
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# DAPI + D-luc
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# DAPI only
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# PBS
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* Let cells sit at room temperature for 10 minutes in the dark to develop signal
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Results/ Conclusions:
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* 2 mL medium is susceptible to drying at outer wells; disrupts cell growth
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* 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.
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* Cells rounded up a bit after sitting in PBS. Next time use 10% FBS in PBS, or try DMEM without red dye.
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* DAPI signal was barely visible
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 +
 
 +
Note: add photos here
 +
 
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----
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'''New cell plates'''<br>
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Dr. Haynes; For more practice staining (Monday)
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* HEK293 luc #4, 6-well plate, ~1x10<sup>5</sup> cells/ well, 4 mL growth medium
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* HEK293 Gal4-EED, 6-well plate, ~1x10<sup>5</sup> cells/ well, 4 mL growth medium
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Stock cultures (T75 flasks)
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* HEK293 luc #4, 1:5
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* HEK293 Gal4-EED, 1:4
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* HEK293 Gal4-EED, 1:4 in 0.5 μg/mL puromycin (to select for Gal4-EED RNAi tuner)
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Current revision

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02/23/13

  • DAPI/ luciferase staining
  • New cell plates

DAPI/ luciferase staining

  • D-luciferin stock solution: 30mg/ml in 1xPBS, working solution is 150ug/ml (200x)
  • Hoescht: made by Dr. Haynes; 5000x in sterile dH2O


  • Diluted Hoescht and D-luc to working concentration (1x) in 1xPBS. Added 2 mL to each well...

Used 3 wells:

  1. DAPI + D-luc
  2. DAPI only
  3. 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


New cell plates
Dr. Haynes; For more practice staining (Monday)

  • HEK293 luc #4, 6-well plate, ~1x105 cells/ well, 4 mL growth medium
  • HEK293 Gal4-EED, 6-well plate, ~1x105 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)




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