User:Karmella Haynes/Notebook/PcTF Genomics/2014/09/10

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09/10/14

  • Lenti Transfection: Day 2 - change out medium (LaBaer lab)



Lenti Transfection: Day 2 - change out medium
Note: At this point, cells are producing virus. Follow extra biosafety precautions.
PPE note: Wear 2x pairs of gloves. Dispose of and replace outer gloves as needed.

  • Medium change 1 - 10:00 am
  1. In the BS cabinet, set up 2x 50 mL conicals of 10 mL of 10x Wescodyne (brown) germicidal detergent. Use one for discarding viral medium, one for rinsing pipettes.
  2. Checked GFP control (sample 7) under scope. Nearly 100% expression. (Note: scope had no RFP filter set)
  3. For each well (one at a time)...
    1. Removed 1.5 mL growth medium, dispensed into Wescodyne, rinsed pipette in Wesc., discarded pipette
    2. Slowly and carefully added 2 mL fresh growth medium, rinsed pipette in Wesc., discarded pipette
    3. Final volume ~3 mL
  4. Placed plates back into 37°C incubator.
  5. Let discarded medium/Wesc. sit for 15 min.
  6. Wiped down BS cabinet surface with Microbiocide.
  7. Aspirated off discarded waste, then rinse solution.


  • Medium change 2 - 4:00 pm
  1. Same biosafety/ discard set-up as before
  2. For each well (one at a time)...
    1. Removed 2.5 mL growth medium, dispensed into Wescodyne, rinsed pipette in Wesc., discarded pipette
    2. Slowly and carefully added 5.5 mL fresh growth medium, rinsed pipette in Wesc., discarded pipette
  3. Placed plates back into 37°C incubator.
  4. Same biosafety/ discard procedure as before



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