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

Haynes Lab - Short Projects

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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