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:
 * 3) spray and wipe the hood surface with 70% ethanol
 * 4) place the following materials into the hood: waste beaker, tube rack, polystyrene tube, pipetting materials, pre-warmed DMEM
 * 5) tape a biohazard bag to the front of the hood
 * 6) Observe the cells under microscope – check for contamination and note cell confluence
 * 7) Place the cells in the hood
 * 8) Remove the cell culture media, and add 2 mL of DMEM (without serum or Pen/Strep)
 * 9) 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
 * 4) Avoid direct contact between FuGENE and the tube wall
 * 5) Tap to mix
 * 6) 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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