Jacobs:Protocol Transfecting NIH 3T3s with GFP-Actin

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  • 3T3 Fibroblasts (seeded in 35 mm dishes or a 6-well plate)
  • FuGENE 6 transfection reagent (Roche Applied Science Cat# 11 815 091 001)
  • pAcGFP1-Actin vector (Clontech Cat.# 632453)
  • DMEM (Invitrogen #11995-065)
  • Water bath
  • Serological Pipets
  • Pipet aid
  • Pipette tips
  • 20 μL, 200 μL, 1000 uL pipetter
  • Microscope
  • Sterile polystyrene tube
  • Timer
  • Waste beaker
  • Biohazard Bag
  • 70% ethanol
  • Kimwipes
  • Markers
  • Gloves


Adding serum-free DMEM to cells:

  1. Heat DMEM to 37oC in a water bath
  2. Prepare the hood:
    1. spray and wipe the hood surface with 70% ethanol
    2. place the following materials into the hood: waste beaker, tube rack, polystyrene tube, pipetting materials, pre-warmed DMEM
    3. tape a biohazard bag to the front of the hood
  3. Observe the cells under microscope – check for contamination and note cell confluence
  4. Place the cells in the hood
  5. Remove the cell culture media, and add 2 mL of DMEM (without serum or Pen/Strep)
  6. Label the dish (initials) and place the dish back in the incubator

Diluting FuGENE 6 with DMEM:

  1. Aseptically place FuGENE 6 and GFP-Actin DNA in the hood
  2. Pipette 91 uL of DMEM into a sterile polystyrene tube
  3. Pipette 9 uL of FuGENE 6 directly into the DMEM
    1. Avoid direct contact between FuGENE and the tube wall
  4. Tap to mix
  5. Incubate for 5 min @ room temp

Forming FuGENE/DNA complex

  1. Add 5.3 uL of pAcGFP1-Actin vector (1 ug) to the dilute FuGENE
  2. Tap to mix
  3. Incubate for 15 min @ room temp
  4. Remove the dish of cells from the incubator
  5. Add the FuGENE/DNA complex to the cells in a drop-wise fashion
  6. Gently swirl dish to mix
  7. Label the dishes (GFP-Actin, date)
  8. Place the cells back in the incubator (add serum containing medium in 6-24 hrs)
  9. Clean the hood and place all material that contacted cells/medium in a biohazard waste bag; clean the waste beaker by adding bleach for ~15 min. before washing



  • History: CMBL – CRJ/JJR Last updated: 8/10/07

or instead, discuss this protocol.