This procedure will take you about an hour. You should plan on an hour and a half, in case of mishaps.

Protocol for Transfection of Cultured Cells

Materials:

- αMEM serum-free media
- Complete media (αMEM with 10% newborn calf serum)
- Complete media (αMEM with 20% newborn calf serum)
- Confluent HEK293 or 3T3 cells
- Plasmid 348 (µg/πl)
- TurboFect Transfection Reagent

Protocol:

1. Subculture confluent HEK293 or 3T3 cells into a new T-25 flask with 1 million cells.
2. Incubate the cells in the 37˚C incubator for 24 hours.
3. After 24 hours mix 5 µg of plasmid 348 DNA with 5ml of αMEM serum-free media and 15 µl of the TurboFect Transfection Reagent. Incubate at room temperature for 15 minutes.
4. Wash cells twice with 10 ml of αMEM serum-free media.
5. Add the αMEM serum-free media containing the plasmid and the TurboFect from step 2 to the washed cells.
6. Incubate the cells in the 37˚C incubator for one hour.
7. Following the incubation add 5 ml of complete media (αMEM with 20% newborn calf serum) to the flask without removing the αMEM serum-free media containing the plasmid and the TurboFect from step 2.
8. Incubate the cells in the 37˚C incubator for 24 hours.
9. Subculture the cells into 24 ml of complete media (αMEM with 10% newborn calf serum) into a six-well plate using 4 ml of cell suspension for each well.
10. Incubate the cells in the 37˚C incubator for 24 hours.
11. After 24 hours make observations using the fluorescent microscope.

Remember: The next step is to come into the lab to practice with the materials, view a demonstration, or to even just ask questions. Also, if you are ready to take the lab final, please inform your instructor. The lab final will be performed by yourself and will be graded with a rubric.

Submission

- Image of your transfected, fluorescing cells
- Image of conclusions from lab notebook