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Fugene Transfection of 293T/COS Cells

Revision as of 14:07, 10 June 2009 by Jpecherer (Talk | contribs) (→‎Protocol)

Revision as of 14:07, 10 June 2009 by Jpecherer (Talk | contribs) (→‎Protocol)

Protocol

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