Haynes Lab:Notebook/Short Projects/2014/09/10: Difference between revisions

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'''Cell culture'''
'''Cell culture'''
* Make fresh growth medium: DMEM, 10% FBS, 1% pen-strep
* Set up 2x 6-well plates for Gal4-VP64 transfections
* Set up 2x 6-well plates for Gal4-VP64 transfections
** 6 wells dox- (3 will be transfected, 3 not) --> 2.5E/well
** 6 wells dox- (3 will be transfected, 3 not) --> 2.5E5/well
** 6 wells dox+ (3 will be transfected, 3 not) --> 2.5E/well
** 6 wells dox+ (3 will be transfected, 3 not) --> 2.5E5/well
# Follow steps 1-9 of [http://openwetware.org/wiki/Haynes:SplittingCells Passaging Adherent Cells] protocol.
# Harvest cells: Follow steps 1-9 of [http://openwetware.org/wiki/Haynes:SplittingCells Passaging Adherent Cells] protocol.<br>Note: cells are ~75% confluent, so at this point you will have ~7.5E5/ mL = ~7.5E6 in 10 mL.
<br>Note: cells are ~75% confluent, so at this point you will have 10 mL of cells (~7.5E5/ mL).
# Calculation 2: How much growth medium used per unique plate? 6 (+1) wells x 4 mL = 28 mL growth medium
#  
# Calculation 3: How many cells used per unique plate? 6 (+) wells x 2.5E5 cells = 1.75E6 cells
# Calculation 4: How much stock volume of harvested cells = Calc 3? 1.75E6 cells / (~7.5E5/ mL) = 2.3 mL
# In a 50 mL conical, add '''25.7 growth medium''' (which is 28 - 2.3)
# Add '''2.3 mL of harvested cells''' to the 50 mL conical.
# Get a 10 mL pipette and mix the cells by pipettin gup and down (>3 times).
# 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


* Also, passage cells 1:5 in T-75 flasks
* Also, passage cells 1:4 in T-75 flasks
# Get a new T-75
# Add 7.5 mL of growth medium to the flask
# Add 2.5 mL of harvested cells to the flask


* Make fresh growth medium: DMEM, 10% FBS, 1% pen-strep




Revision as of 09:16, 10 September 2014

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09/10/14 - Jonah Thomas

  • Cell culture - Gal4-EED/luc for luciferase activation experiment
  • Passage cells 1:5 (T-75 flasks)


Cell culture

  • Make fresh growth medium: DMEM, 10% FBS, 1% pen-strep
  • Set up 2x 6-well plates for Gal4-VP64 transfections
    • 6 wells dox- (3 will be transfected, 3 not) --> 2.5E5/well
    • 6 wells dox+ (3 will be transfected, 3 not) --> 2.5E5/well
  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. Calculation 2: How much growth medium used per unique plate? 6 (+1) wells x 4 mL = 28 mL growth medium
  3. Calculation 3: How many cells used per unique plate? 6 (+) wells x 2.5E5 cells = 1.75E6 cells
  4. Calculation 4: How much stock volume of harvested cells = Calc 3? 1.75E6 cells / (~7.5E5/ mL) = 2.3 mL
  5. In a 50 mL conical, add 25.7 growth medium (which is 28 - 2.3)
  6. Add 2.3 mL of harvested cells to the 50 mL conical.
  7. Get a 10 mL pipette and mix the cells by pipettin gup and down (>3 times).
  8. 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

  • Also, passage cells 1:4 in T-75 flasks
  1. Get a new T-75
  2. Add 7.5 mL of growth medium to the flask
  3. Add 2.5 mL of harvested cells to the flask




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