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Optimization of Plasmid DNA Transfection
With any transfection procedure, a critical first step is to optimize the transfection conditions. Every cell type and transfection procedure has a characteristic set of requirements for optimal introduction of foreign DNA, and these conditions have a large degree of variability even among cell types that are very similar to one another.
The single most important factor in optimizing transfection efficiency is selecting the proper transfection protocol for the cell type. Once the appropriate transfection method is selected, a transient reporter assay system can be used to optimize the procedure by transfecting a reporter gene under a variety of conditions and monitoring the transfection efficiency by assaying for the reporter gene product.
This section provides general guidelines for optimizing calcium phosphate-mediated gene transfer, electroporation using the Neon Transfection System, and cationic lipid transfection.
Which Invitrogen plasmid DNA transfection reagent product is right for you?
| High-efficiency, versatile reagent for a wide range of common cell types Lipofectamine 2000 | Most efficient versatile reagent for the widest range of cell types including difficult-to-transfect cells Lipofectamine 3000 | High-efficiency electroporation for all cell lines; best for primary & stem cells Neon Transfection System | |
|---|---|---|---|
| Top-selling product (Cat. No.) | 1.5 mL (11668-019) | 1.5 mL (L3000-015) | Starter pack (MPK5000S) |
| Sample type |
|
|
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| Transfection efficiency | High | Superior | Maximal |
| Cell viability | High | Superior | Good |
| Protocol | Download protocol | Download protocol | Download protocol |
| Order now | Order now | Order now |
Considerations for calcium phosphate co-precipitation
The primary factors that influence the efficiency of calcium phosphate transfection are the amount of DNA in the calcium-phosphate-DNA co-precipitate, the length of time the cell is incubated with the co-precipitate, and the use and duration of glycerol or DMSO shock.
The total DNA amount used in calcium phosphate transfection is usually 10–50 μg in 450 μL sterile water and 50 μL of 2.5 M CaCl2 per 10 cm dish, but varies widely among plasmid preparations as well as with different cells and media. While with some cell lines 10–15 μg of DNA added to a 10 cm dish results in excessive cell death and very little uptake of DNA, for other cell lines, especially primary cells, much higher concentrations of DNA are required. Each new plasmid preparation and each new cell line being transfected should be tested for optimum DNA concentration.
The optimal length of time that the cells are incubated with co-precipitate also varies with cell type. Some hardy cell types, such as HeLa, NIH 3T3, and BALB/c 3T3, are efficiently transfected by leaving the co-precipitate on for up to 16 hours, which might kill some more sensitive cells.
A pilot experiment varying the amount of DNA, incubation time, and exposure to glycerol or DMSO shock will indicate whether the cell type is tolerant to long exposure to a calcium phosphate precipitate and whether glycerol shock should be used. Once the results of the pilot experiment are obtained, further optimization can be performed by adjusting the experimental variables even finer. For instance, if shocking the cells with 10% glycerol for 3 minutes as shown in the example below enhances transfection efficiency, an experiment varying the time of glycerol shock or using 10–20% DMSO shock might also be tried.
Table 1. Pilot experiment example for the optimization of transfection by calcium phosphate co-precipitation
| Dish (10 cm) | Reporter plasmid (μg) | Incubation (hr) | Glycerol shock (min) |
|---|---|---|---|
| 1 | 5 | 6 | - |
| 2 | 10 | 6 | - |
| 3 | 15 | 6 | - |
| 4 | 20 | 16 | - |
| 5 | 25 | 16 | - |
| 6 | 30 | 16 | - |
| 7 | 5 | 6 | 3 |
| 8 | 10 | 6 | 3 |
| 9 | 15 | 6 | 3 |
| 10 | 20 | 16 | 3 |
| 11 | 25 | 16 | 3 |
| 12 | 30 | 16 | 3 |
Considerations for cationic lipid-mediated delivery
Four primary parameters affect the success of DNA transfection by cationic liposomes: the amount of DNA, the ratio of transfection reagent to DNA, incubation time of the lipid-DNA complex, and the cell density at the time of complex addition. These factors should be systematically examined for every cell type and vector combination, and once optimized, kept constant in all future experiments to help ensure reproducible results.
For best results, follow the optimization protocols provided by the manufacturers of the reagent.
The optimal amount of DNA varies depending on the characteristics of the transfected plasmid (e.g., promoter, size of plasmid, origin of replication), number of cells to be transfected, size of the culture dish, and the target cell line used. In many of the cell types tested, relatively small amounts of DNA are effectively taken up and expressed. In fact, higher levels of DNA can be inhibitory in some cell types with certain cationic lipid preparations. In addition, cytotoxicity may result if a plasmid encoding a toxic protein or too much plasmid with a high expression rate is used.
The overall charge of the transfection complexes is determined by the ratio of transfection reagent to DNA. The negative charge contributed by phosphates within the DNA backbone needs to be offset by the positive charge contributed from the transfection reagent for both good complex formation and for neutralizing the electrostatic repulsion imparted on the DNA by the negatively charged cell membrane.
The optimal ratio of transfection reagent to DNA is highly cell type-dependent. As a starting point, the amount of transfection reagent should be varied while keeping a constant plasmid DNA concentration (for example, 1:1, 3:1, and 5:1 ratios of volume to mass). Additional benefits may be derived by maintaining the ratio and increasing the amount of plasmid added.
The optimal incubation period of cells with the transfection complexes depends on the cell line and transfection reagent used. In general, transfection efficiency increases with time of exposure to the lipid reagent-DNA complex, although toxic conditions can develop with prolonged exposure to certain lipid reagents, requiring removal by centrifugation or dilution with fresh medium after a given incubation period to minimize cytotoxic effects. However, newer and gentler transfection reagents such as Lipofectamine 3000 reagent do not necessitate complex removal or dilution after transfection.
When using cationic lipid reagents that require adding or replacing the medium, vary the incubation time after complex addition (e.g., 30 minutes to 4 hours, or even overnight) and monitor cell morphology during this interval, particularly if the cells are maintained in serum-free medium as some cell lines lose viability under these conditions.
Cell density also affects overall transfection efficiency. To achieve transcription and ultimately protein production, nuclear deposition of DNA is required, which is largely dependent on membrane dissolution and reformation during mitosis, requiring that the cells have to be actively dividing.
For adherent cells, the best efficiency is often attained at a confluency of 80%, but protocol recommendations may range from 40–90%. For suspension cells, we recommend splitting the cells the day prior to transfection to ensure that the cells will be in optimal physiological condition for the transfection procedure. The optimal density is highly dependent on cell type and reagent-specific toxicity and should be determined empirically.
Figure 1. Example transfection workflow using the Lipofectamine 3000 transfection reagent.
Considerations for electroporation
Electroporation is mainly dependent on the combination of three electric parameters: the pulse voltage, pulse width, and pulse number. Perhaps because it is not a chemically based protocol, electroporation is less affected by DNA concentration; however, it requires almost five-fold more cells and DNA compared to calcium phosphate-mediated transfection. Generally, 1–5 μg of DNA per 107 cells is sufficient, and there is a good linear correlation between the amount of DNA present and the amount taken up.
The objective in optimizing electroporation parameters is to find a pulse that maintains 40–80% survival of the cells. The pulse width is determined by the capacitance of the power source and the extent to which this can be varied depends on the electronics of the power supply generating the pulse. If excessive cell death occurs, the length of the pulse can be lowered by lowering the capacitance.
Keeping cells on ice often improves cell viability and results in higher effective transfection frequency, especially at high power which can lead to heating [1]. However, some cell lines electroporate with higher efficiency at room temperature under low voltage/high capacitance conditions [2].
The Neon Transfection System, available from Thermo Fisher Scientific, is pre-programmed with 18- and 24-well optimization protocols that allow quick optimization of electric parameters for many adherent and suspension cell lines within days. Cell line-specific optimized protocols for the Neon Transfection System can also be conveniently downloaded to maximize transfection efficiencies for many commonly used cell types.
Best practices for DNA transfection
1) Start with healthy cells
a. Passage cells 3–4 times after thawing before using them in transfection experiments. This gives the cells time to recover from the thawing procedure and return to their normal rate of growth.
b. Only use cells >90% viability—you can easily determine cell viability using trypan blue stain.
c. Passage cells on a regular basis and do not allow the cells to become confluent or overgrown. Allowing cells to become confluent may change their growth rate as well as cell morphology.
- Passage cells at or before 90% confluency. We recommend using Gibco TrypLE reagent for detachment of cells. TrypLE reagent can be stored in the hood at room temperature, and you can skip the PBS wash by adding extra TrypLE reagent and aspirating all but enough solution to cover the surface of the flask.
- Passage conditions are dependent on the individual cell line. Some rules of thumb:
- For fast-growing cells, with a doubling time of every 16 hr (e.g., HEK-293), split cells 1:10
- For slow-growing cells, with a doubling time of every 36 hr (e.g., primary cells), split cells 1:5
d. Maintain frozen stocks of cell lines and regularly thaw new cells. Cells can change growth rate and morphology at high passage numbers (>30–40).
2) Plan your experiment before going into the lab
It is important to familiarize yourself with the protocol, determine how much material you need, and confirm that you have everything you need before getting started.
a. Start by designing a plate map outlining each treatment or experimental condition.
b. Calculate the amount of lipid and DNA stock you will need, and confirm that you have enough of the following materials: Gibco Opti-MEM medium, Invitrogen Lipofectamine Reagent, and DNA (0.5–1 µg/µL).
3) Use high-quality DNA in your transfection
a. Prepare DNA using an endotoxin-free kit or protocol.
b. Determine the DNA purity by measuring the OD 260/280 ratio, which should be between 1.7–1.9. Higher or lower ratios indicate impurities and should not be used in transfection experiments.
c. Dilute DNA in DNase/RNase-free water or TE.
d. Prepare working concentrations of 0.5–1 µg/µL. DNA can be concentrated by Thermo Scientific SpeedVac concentrator or diafiltration.
4) Plate your cells the day before you plan to transfect
a. If plated >1 day prior to transfection, the transfection efficiency may decrease.
b. Seed your cells at a density such that they will be 70–90% confluent at the time of transfection.
c. Cell density at the time of transfection affects the transfection efficiency. To simplify transfection optimization, or to save time, we recommend plating at 2 different densities to ensure a high transfection efficiency.
d. Cell plating and transfection can be performed at the same time or by using a reverse transfection protocol. In a reverse transfection, you need to use 2.5 times more cells than you would use in a regular or forward transfection.
e. Transfection complexes can be added to cells in media containing antibiotics and serum without impacting the transfection efficiency.
5) Prepare lipid DNA complexes
a. For maximum performance, we recommend complexing the lipid and DNA in Opti-MEM medium. Alternatively, a serum-free medium can be used.
b. Dilute the lipid in Opti-MEM medium. Dilute the DNA in a second tube of Opti-MEM medium, and mix equal volumes to complex the lipid and DNA. The 2-step dilution method results in higher quality data and generates more reproducible results compared to adding the lipid directly to the diluted DNA.
c. Once the lipid is diluted in Opti-MEM medium, the optimal incubation time is 2 to 5 min before adding the diluted DNA. Do not incubate the diluted lipid for longer than 20 min.
d. The DNA Opti-MEM solution is more stable and can be prepared up to 4 hr in advance but not longer.
e. Once equal volumes of the diluted lipid and DNA have been combined, mix the solution by pipetting up and down slowly, flicking the bottom of the tube, or vortexing quickly.
f. After the lipid DNA complex has incubated for 5 to 10 min, transfer the complex to the well containing cells and growth medium. Add complexes drop-wise on top of the medium and gently rock the plate after adding to the mix. Avoid forceful dispensing of lipid DNA complex in the well because it may displace cells.
6) Use a positive control such as GFP or LacZ reporter plasmid to assess transfection efficiency
The number or percent of cells transfected and the intensity of GFP expression can easily be determined using a fluorescence microscopy (e.g., EVOS microscope or flow cytometry (e.g., an Attune flow cytometer)). LacZ expression can be determined using commercially available kits.
a. Plasmid expression should be visible or detectable 24 to 48 hrs post-transfection.
b. The positive control can be in a separate well or co-transfected with your plasmid of interest. For co-transfection, you can mix 50–100 ng of GFP plasmid with 100 ng of your plasmid of interest together in Opti-MEM medium prior to adding the diluted lipid.
1) Start with healthy cells
a. Passage cells 3–4 times after thawing before using them in transfection experiments. This gives the cells time to recover from the thawing procedure and return to their normal rate of growth.
b. Only use cells >90% viability—you can easily determine cell viability using trypan blue stain.
c. Passage cells on a regular basis and do not allow the cells to become confluent or overgrown. Allowing cells to become confluent may change their growth rate as well as cell morphology.
- Passage cells at or before 90% confluency. We recommend using Gibco TrypLE reagent for detachment of cells. TrypLE reagent can be stored in the hood at room temperature, and you can skip the PBS wash by adding extra TrypLE reagent and aspirating all but enough solution to cover the surface of the flask.
- Passage conditions are dependent on the individual cell line. Some rules of thumb:
- For fast-growing cells, with a doubling time of every 16 hr (e.g., HEK-293), split cells 1:10
- For slow-growing cells, with a doubling time of every 36 hr (e.g., primary cells), split cells 1:5
d. Maintain frozen stocks of cell lines and regularly thaw new cells. Cells can change growth rate and morphology at high passage numbers (>30–40).
2) Plan your experiment before going into the lab
It is important to familiarize yourself with the protocol, determine how much material you need, and confirm that you have everything you need before getting started.
a. Start by designing a plate map outlining each treatment or experimental condition.
b. Calculate the amount of lipid and DNA stock you will need, and confirm that you have enough of the following materials: Gibco Opti-MEM medium, Invitrogen Lipofectamine Reagent, and DNA (0.5–1 µg/µL).
3) Use high-quality DNA in your transfection
a. Prepare DNA using an endotoxin-free kit or protocol.
b. Determine the DNA purity by measuring the OD 260/280 ratio, which should be between 1.7–1.9. Higher or lower ratios indicate impurities and should not be used in transfection experiments.
c. Dilute DNA in DNase/RNase-free water or TE.
d. Prepare working concentrations of 0.5–1 µg/µL. DNA can be concentrated by Thermo Scientific SpeedVac concentrator or diafiltration.
4) Plate your cells the day before you plan to transfect
a. If plated >1 day prior to transfection, the transfection efficiency may decrease.
b. Seed your cells at a density such that they will be 70–90% confluent at the time of transfection.
c. Cell density at the time of transfection affects the transfection efficiency. To simplify transfection optimization, or to save time, we recommend plating at 2 different densities to ensure a high transfection efficiency.
d. Cell plating and transfection can be performed at the same time or by using a reverse transfection protocol. In a reverse transfection, you need to use 2.5 times more cells than you would use in a regular or forward transfection.
e. Transfection complexes can be added to cells in media containing antibiotics and serum without impacting the transfection efficiency.
5) Prepare lipid DNA complexes
a. For maximum performance, we recommend complexing the lipid and DNA in Opti-MEM medium. Alternatively, a serum-free medium can be used.
b. Dilute the lipid in Opti-MEM medium. Dilute the DNA in a second tube of Opti-MEM medium, and mix equal volumes to complex the lipid and DNA. The 2-step dilution method results in higher quality data and generates more reproducible results compared to adding the lipid directly to the diluted DNA.
c. Once the lipid is diluted in Opti-MEM medium, the optimal incubation time is 2 to 5 min before adding the diluted DNA. Do not incubate the diluted lipid for longer than 20 min.
d. The DNA Opti-MEM solution is more stable and can be prepared up to 4 hr in advance but not longer.
e. Once equal volumes of the diluted lipid and DNA have been combined, mix the solution by pipetting up and down slowly, flicking the bottom of the tube, or vortexing quickly.
f. After the lipid DNA complex has incubated for 5 to 10 min, transfer the complex to the well containing cells and growth medium. Add complexes drop-wise on top of the medium and gently rock the plate after adding to the mix. Avoid forceful dispensing of lipid DNA complex in the well because it may displace cells.
6) Use a positive control such as GFP or LacZ reporter plasmid to assess transfection efficiency
The number or percent of cells transfected and the intensity of GFP expression can easily be determined using a fluorescence microscopy (e.g., EVOS microscope or flow cytometry (e.g., an Attune flow cytometer)). LacZ expression can be determined using commercially available kits.
a. Plasmid expression should be visible or detectable 24 to 48 hrs post-transfection.
b. The positive control can be in a separate well or co-transfected with your plasmid of interest. For co-transfection, you can mix 50–100 ng of GFP plasmid with 100 ng of your plasmid of interest together in Opti-MEM medium prior to adding the diluted lipid.
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References
- Potter H, Weir L, Leder L. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984;81(22):7161–7165. doi: 10.1073/pnas.81.22.7161.
- Chu G, Hayakawa H, Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987; 15(3): 1311–1326. doi: 10.1093/nar/15.3.1311.
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