Haynes Lab:Notebook/Short Projects/2014/09/10

Haynes Lab - Short Projects

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09/10/14 - Jonah Thomas Cell culture - Gal4-EED/luc for luciferase activation experiment Passage cells 1:4 (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 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. 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 pipetting up 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 1:4 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