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

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  • 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)