Lipofectamine 3000 Adherent Transfection Protocol

  1. Dilute 1uL of Lipofectamine in 25uL of OptiMEM/DMEM
  2. Dilute 1ug of DNA in 50uL of OptiMEM/DMEM
  3. Add 2uL of P3000 Reagent
  4. Add 25uL of the DNA-P3000 mix to 25uL of the diluted Lipofectamine
  5. Incubate for 5 minutes
  6. Add 50uL of the DNA-Lipofectamine-P3000 mix to each well

 

Lipofectamine 3000 Suspended Transfection Protocol

  1. Follow DNA-lipid complexing steps as in adherent transfection before preparing cells.
  2. To prepare cells, begin with a confluent plate of cells. Wash with 3mL of PBS. Add 2.5mL of trypsin
  3. Wait 5 minutes or until the cells have unadhered.
  4. Quench trypsin with 10mL of Complete Media. Transfer to 50mL conical.
  5. Centrifuge at 2,500 rpm for 5 minutes. Aspirate supernatant.
  6. Resuspend cells in 10mL DMEM.
  7. Add cells to 24-well plate at a density of 150,000 - 200,000 cells in 500uL DMEM per well.
  8. Add DNA-lipid complexes to wells. 
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