Bio-Rad TransFectin™ Lipid Reagent - Manual (Page 2)

Table 1. Suggested Reagent Quantities for Different Sizes of Plates/Wells
Culture Surface Volume of Plasmid Volume of Serum TransFectin
Vessel Size Area (cm 2 ) Plating Media DNA Free Medium Reagent
96 well 0.32 0.1 ml 50–200 ng 25 µl 0.1–0.6 µl
24 well 1.9 0.5 ml 0.25–1.0 µg 50 µl 0.25–4.0 µl
12 well 3.8 1.0 ml 0.50–2.0 µg 100 µl 2.0–8.0 µl
6 well/35mm 9.2 2.0 ml 1.0– 4.0 µg 250 µl 5.0–15 µl
60 mm 21 5.0 ml 2.0–8.0 µg 500 µl 15–20 µl
100 mm 60 10.0 ml 12–36 µg 1.5 ml 40–60 µl
Protocol for Transfection of Adherent Cells
(24 Well Plates)
1. The day before transfection, inoculate 24-well plates with an appropriate number of cells in
serum-containing medium such that they will be 50 to 90% confluent the following day. For most cell
lines, plating 0.5 to 8.0 x 10
5 cells in 0.5 ml of medium should be appropriate. Incubate the cells at
37°C in a 5% CO
2
incubator overnight.
2. For each well, prepare plasmid in 50 µl of serum-free medium. Use 0.25 to 1.0 µg of plasmid DNA for
each 50 µl of serum-free medium.
3. Prepare the TransFectin reagent in 50 µl of serum-free medium. Use 0.25 to 4.0 µl for each well as a
starting point for optimizing the reaction.
4. Mix the DNA and TransFectin solutions together. Gently mix by tapping or pipetting. Incubate 20 minutes
at room temperature.
5. Add 100 µl of the DNA–TransFectin complexes directly to cells in serum-containing medium. Swirl
gently. Incubate the cells at 37°C in a 5% CO
2
incubator. Additional medium may be added 4 to 6 hours
after addition of complexes.
6. For transient expression, assay for reporter gene activity 24 to 48 hours after the transfection.
7. For stable expression of the transfected plasmid sequence, remove transfection medium 24 hours after
transfection and trypsinize the cells. Transfer the cells to a fresh plate with growth medium containing no
selective agent. The following day, replace with new medium containing the selective agent. Continue
incubating for 1 to 2 weeks to allow growth of the cells expressing the transgene.
Protocol for Transfection of Suspension Cells
(24 Well Plates)
1. The day before transfection, dilute the cells such that they will be in log phase growth the following day.
For most cell lines, inoculating 0.75 to 8.0 x 10 5 cells per 0.5 ml of medium should be appropriate.
Incubate the cells overnight at 37°C in a 5% CO
2
incubator.
2. For each well, prepare plasmid in 50 µl of serum-free medium. Use 0.25 to 1.0 µg of plasmid for each
50 µl of medium.
3. Prepare TransFectin in 50 µl of serum-free medium for each well. Use 0.25 to 4.0 µl of lipid for each 50 µl
of medium.
4. Mix the plasmid and TransFectin solutions together. Gently mix by tapping or pipetting. Incubate 20
minutes at room temperature.
5. Add the 100 µl of DNA–TransFectin complexes to the cell suspension; rock the plate gently to ensure
adequate mixing of the solutions.
6. Incubate the cells at 37°C in a 5% CO
2
incubator. Additional medium may be added 4 to 6 hours after
addition of complexes.
7. For transient expression, assay reporter gene activity 24–48 hours after transfection.
4106254 Rev A
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