Haynes Lab:Notebook/Short Projects/2014/09/22

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09/22/14 - Jonah Thomas

  • Cell culture - Gal4-EED/luc for luciferase activation experiment
  • Passage cells 1:4 (T-75 flasks)

Cell culture

  • Set up 2x 6-well plates for Gal4-VP64 transfections
  1. Harvest cells: Follow steps 1-9 of Passaging Adherent Cells protocol.
    Note: cells are ~75% confluent, so at this point you will have ~7.5E5/ mL = ~7.5E6 in 10 mL.
  2. 28 mL of growth medium and harvested cells was aliquot into a 50mL conical vial (1 well extra was added to account for pipetting errors)
    1. 2.3 mL of harvested cells were added to the conical vial (this is equal to ~1.75E6 cells)
    2. 25.7 mL of growth medium was added to the conical vial
  3. Get a 10 mL pipette and mix the cells by pipetting up and down (>3 times).
  4. Draw up and dispense 4 mL of the suspended cells into each well in the 6-well plate.

Note: Do this whole procedure separately for the dox- and the dox+ cells Note: For the dox+ plate, 4μL of 1mg/mL doxycycline was added to each well (1μL was added for each mL of the unique well's volume = 4mL)

  • A 1:4 passage of the cells was also performed into a T-75 flask