This protocol is for transfecting plasmid DNA into mammalian cells.
Protocol 1) Stable transfection using Lipofectamine 2000 in 6 well plates
- Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Generally need to have 2 µg of DNA for each tranfection. Purify the linear fragment using any appropriate method.
- Day 1) Plate cells in 6 well plates in 2 ml media (can have pen/strep). Plate an amount that will result in approximately 85 to 90% confluence (see table below) on the day of transfection.
- Day 2) Prepare transfection cocktail. In Tube "A" place 2 µg of DNA in Opti-Mem-I for a final volume of 200 µl.
- In tube "B" place 190 µl of Opti-Mem-I and 10 µl Lipofectamine 2000. Incubate for 5 minutes at room temperature, but not more than 10 minutes
- Gently combine tubes A and B and incubate at least 20 minutes, but can be up to several hours.
- Remove media from 6-well plates and add 1.6 ml of fresh media (which can contain pen/strep).
- Add the lipofectamine/DNA mixture to each well and gently swirl to mix.
- Incubate at 37°C for 4 to 6 hours for delicate cells or overnight for tough cells.
- After incubation, remove media and replace with fresh media. If the transfected DNA contains a fluorescent protein, check for transfection efficiency by viewing in a fluorescent microscope. Allow cells to grow for 1 to 2 days.
- Trypsinize cells and transfer to a 10 cm plate and add selecting antibiotic
Option A) Transient transfection
- For a transient transfection follow Protocol 1) except you don't have to cut the plasmid and do not add any selecting antibiotic. Assays should be done immediately following transfection
Option B) 24-well plates
- Seed number of cells that will result in 90%
Option B) 24-well plates using Lipofectamine LTX
- Use number of cells shown below, reduce media volume to 500 µl/well,
- In tube "A" place 500 ng of DNA in Opti-Mem-I to a final volume of 50 µl
- In tube "B" place 3 µl Lipofectamine LTX in 47 µl of Opti-Mem-I. Note, can adjust the amount of Lipofectamine LTX and DNA. Incubate 5 minutes at room temperature, but not more than 10 min.
- Optional: Add 0.4 µg of the PLUS reagent per well.
- Gently mix tube A and B and incubate at least 20 minutes but can be several hours at room temp.
- Remove media from 24 well plates and add 400 µl of fresh media, which can include pen/strep.
- Add the lipofectamine/DNA mixture to 24 well plates and gently swirl to mix
- Incubate in tissue culture incubator for 4-6 hours or overnight.
Cell numbers to plate the day before transfecting