User:Karmella Haynes/Notebook/PcTF Genomics/2014/11/15

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11/15/14

  • Transfection: KAH126/MV2 into U2OS



Transfection: KAH126/MV2 into U2OS
Strategy: instead of trying to increase transfection efficiency, do a "quasi-subcloning" where transfected cells are separated into smaller wells, and wells showing highest RFP will be selected, pooled, and re-plated.

  • 6-well plate seeded on 11/14/14 (~2.5E5 cells/well)
  • Change medium to p/s-free before transfection (4 mL per well)
  • Use standard Lipofectamine LTX procedure w/ 2000 ng plasmid
  1. KAH126/MV2
  2. mock
  3. no transfection
  • Grow overnight, look for RFP next day
  • Next day: harvest transfected cells and use 1/2 (5.0E5) for 2 different plates
    • 24-well plate - ~2.1E4 cells/well
    • 96-well plate - ~5.2E4 cells/well



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