Lipofectamine

Submitted by kdorfman on Tue, 09/25/2012 - 17:56

Lipofectamine2000 Plasmid Transfection

24-48 hrs before class,

  • transfect cells (at 40-60% confluency)
  • Medium without
    • antibiotic
    • serum
    • L-glutamine

Protocol

  • Dilute DNA with DMEM
  • Dilute lipofectamine2000 with DMEM, incubate <5 min at RT
  • Mix DNA with Lipofectamine2000, incubate >15 min at RT
  • Rinse 2x with DMEM
  • Add complexes to cells, incubate 4 hr 37C
  • Rinse 2x with warm PBS

  • Add normal medium back to cells

  • Per 35 mm dish (1 coverslip):

    • 5 µg DNA in 500 µL
    • 10 µL lipofectamine in 500 µL