Bio-Rad TransFectin Lipid Reagent User Manual
TransFectin™Lipid Reagent
0.5 ml 170-3350
1.0 ml 170-3351
5 x 1.0 ml 170-3352
For Research Use Only
Store at 4°C
Storage and Stability
TransFectin Lipid Reagent is shipped on ice. Store at 4º C upon receipt. Do not store below 0ºC. TransFectin is
stable for 6 months from date of purchase when stored at 4º C.
Contents
TransFectin contains either 0.5 ml (Catalog Number 170-3350), 1.0 ml (Catalog Number 170-3551), or 5 x 1.0 ml
(Catalog Number 170-3352) of a proprietary lipid formulation. One milliliter is generally sufficient for 125 to 200 cell
transfections in 35 mm plates. TransFectin comes as a 1.5 mg/ml solution.
Reagent
TransFectin Lipid Reagent is a mixture of a proprietary cationic compound and a co-lipid (1,2-Dioleoyl-sn-Glycero-3Phosphoethanolamine). These compounds have been optimized for intracellular delivery of nucleic acids into cultured
mammalian cells in the presence of serum at cell densities from 50% to greater than 90%. For most cell
lines, high levels of expression can be obtained using concentrations of TransFectin and DNA suggested in this manual
for given sizes of plates/wells. If presently using a cationic lipid for transfection, start using TransFectin at current
conditions and also try reducing and increasing the volume of TransFectin from 25 to 50% of current amount. For best
results it is important to determine the optimal amount of DNA and lipid for any given cell line.
Recommendations for Best Results
• For most cell lines use a ratio of DNA (µg) to lipid (µl) of 1:2–1:3 as a starting point to optimize conditions.
• Invert the tube to mix contents before using.
• TransFectin is designed to work in media containing serum but can be used in the absence of serum.
• Use sterile polystyrene plastic ware (e.g. 12 x 75 mm tubes or multi-well trays) to prepare the plasmid solutions
and lipid solutions. Polystyrene is recommended because cationic lipid-plasmid complexes may bind to
polypropylene.
Optimization
Determining the optimum conditions for transfection efficiency is essential for maximizing gene expression
and minimizing cellular toxicity. The two most important parameters to optimize for any given culture vessel
and cell density are the concentration of TransFectin and the amount of nucleic acid. See Table 1 for
suggested reagent and DNA concentrations for different culture vessel sizes. In general,
gene expression will increase, plateau and then decrease with increasing concentrations of TransFectin.
This decrease in gene expression correlates with reduced cell viability. As increasing amounts of plasmid
are added to the cells, gene expression will increase, then plateau or even decrease since the amount of
DNA used can directly affect toxicity. If toxicity is encountered, try reducing lipid or DNA amounts used in
transfection. The concentration of TransFectin and the amount of plasmid required for maximal expression
may vary from one cell line to another.
Table 1. Suggested Reagent Quantities for Different Sizes of Plates/Wells
Culture Surface Volume of Plasmid Volume of Serum TransFectin
Vessel Size Area (cm2) 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% CO2incubator 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% CO2incubator. 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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