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Fugene Transfection of 293T/COS Cells

Revision as of 14:34, 8 June 2009 by Davebridges (Talk | contribs) (wrote initial page)

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Revision as of 14:34, 8 June 2009 by Davebridges (Talk | contribs) (wrote initial page)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Protocol

  1. Warm OptiMEM, COS-FBS Media and PBS -/-.
  2. Calculate amount of Fugene needed.
  • Per ug of DNA need 3 uL Fugene.
  • Per uL of Fugene need 16 uL of OptiMEM.
  1. Add Fugene to OptiMEM, incubate ~5 min.
  2. Add required amount of DNA to eppendorf tubes.
  3. Add 51 uL/ug OptiMEM/Fugene to each DNA tube.
  4. Incubat 5-20 min.
  5. Split confluent cells 2-3X into fresh dishes as follows:
  6. Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
  7. Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
  8. Add 25 mL COS/FBS
  9. Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
  10. Add DNA/Fugene/DMEM to cells
  11. Leave mixture on Cells for 24-48h to allow protein to accumulate
Last modified on 29 August 2011, at 13:22