Haynes Lab:Notebook/Short Projects/2015/03/01

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Karmella Haynes - 03/01/15

  • Passaging - KAH126, split 1:10
  • 6-well plate - KAH126
  • Dox induction - KAH126

Passaging - KAH126

  • Protocol: Splitting Cells
  • Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S
  • Use 90-100% confluent T-75
  • Make 1 new T-75 at a 1:10 dilution
  • Passage number: 2



6-well plate - KAH126

  • Use same resuspension from old flask in Passaging - KAH126
  • Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration
  • Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium
  • Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well
  • Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well
  • Make a batch of diluted cells for new 6-well plate:
    • Into a sterile 50 mL conical add...
    • (6 wells + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells
    • (6 wells + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium



Dox induction - KAH126

  • Induce cells in 6-well plate with dox to express PcTF (RFP)
  • Stock concentration of dox = 1.0 mg/mL
  • Desired final concentration of dox = 1.0 μg/mL (1000x diluted)
    • Therefore add 1.0 μL of stock dox for every 1.0 mL medium in well
  • 4.0 mL medium in well x 1.0 μL stock dox = 4.0 μL stock dox to be added to each well



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