Jacobs:Protocol Transfecting NIH 3T3s with GFP-Actin

Materials

• 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

Procedure

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

Contact

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

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