QuickFectin™ Transfection Reagent
Quickfectin™ is a new generation transfection reagent optimized for routine DNA transfection in mammalian cells using a quick, simple procedure. Quickfectin™ is specially formulated for both transient and stable transfection in various human embryonic kidney 293 cell lines, including 293T, FreeStyle, 293FT, 293A, 293TA, 293TN and 293E. The proprietary formulation ensures consistently low cytotoxicity and high transfection efficiency (typically >95%) in the presence or absence of serum and antibiotics/antimycotics. Quickfectin™ is ideally suited for large-scale transfection of adherent/suspension 293 cells and for high-throughput protein/virus production. Quickfectin™ has been tested to be highly effective in the delivery of gene clones for overexpression and the production of recombinant proteins and high titer viral particles, such as retrovirus, lentivirus, and AAV. Many common cell lines, including CHO, HeLa, COS-7, 3T3 mouse fibroblasts, can also be successfully transfected with Quickfectin™.
Reagents Supplied:
Quickfectin™ Transfection Reagent, aliquot in 1.0 ml/vial, each vial is sufficient for transfection of at least 300 μg of DNA using the standard protocol.
Quality Control Assays:
Quickfectin™ is lot tested against standard 293 cell lines (in both adherent and suspension) to assure consistent transfection efficiency and performance.
Product Storage:
The product is shipped at ambient temperature. Upon arrival, store the product at 4°C or lower for long-term storage (DO NOT FREEZE). If stored properly, the product will be stable for 12 months or longer.
FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.
Notice to purchaser: Quickfectin™ transfection reagent are developed and sold exclusively for research purposes. Neither the products, nor any individual components, have been tested for use in humans or animals.
The efficient transfection of clone genes into mammalian cells plays a critical role in the gene overexpression for functional analysis and in the production of recombinant proteins and viral particles. To establish a simple and scalable transfection system, we have developed a new generation of transfection reagent - Quickfectin™ (Qfectin) and studied transient transfection in both adherent and serum-free suspension 293 cells. We have used quantification of green fluorescent protein (GFP) to monitor transfection efficiency (%GFP+) and protein expression level (MFI). As shown in the Figure below, Quickfectin™ delivers excellent transfection efficiency and expression level that are superior to the industry’s leading reagent.
Parameters including Quickfectin™/DNA concentration and ratio, incubation time for the complex formation, and cell confluence or density at the time of transfection were optimized (see Protocol for more details). Over 97% of GFP-positive 293 cells were obtained with Quickfectin™ in both adherent and suspension 293 cells (see the data example above in suspension 293 cells). Both transfection efficiency and expression level outperform the current industry’s leading brand Fugene® under described optimal conditions. Scale-up of the Quickfectin™ transfection system to 10 liter resulted in similarly high transfection efficiency. Quickfectin™ has been routinely used for the production of large quantities of recombinant proteins and viral particles using both adherent and serum-free suspension mammalian cells.
Quickfectin™ transfection reaction can be conveniently performed within 5 minutes using a simple and fast 3-step procedure below:
Step 1: Dilute endotoxin-free DNA with sterile PBS (pH7.4) or any growth medium to 20-50 ng/μL
Note: if the DNA concentration is below 50 ng/uL, it can be directly used for the following reaction.
Step2: Add Quickfectin™ to the above diluted DNA at a ratio of 3 (i.,e., 3 μL Quickfectin™ per μg of DNA) or higher (up to 9) and incubate at room temperature for 5 minutes.
Step3: Add Quickfectin™-DNA reaction mixture dropwise to the cells and gently mix to distribute the complex evenly and incubate for 24 - 72 hrs before analysis of expression.
Several standard protocols are listed below for experiments performed in 12-well plates optimized for adherent or suspension 293 cells. If using other cell type or culture plates/flasks, optimize transfection condition carefully and multiply the suggested Quickfectin™/DNA quantities by the relative surface area or volume (see Table 1 for more details).
Protocol I: Standard Protocol for transient transfection (adherent 293 cells)
1. Cell plating:
a) On the day before transfection, plate 1 ml of cells at a density of 2 - 5 x 105 cells/mL in complete growth medium per well in a 12-well plate to obtain 50 - 70% confluence on the following day of transfection.
b) Incubate cells overnight
2. Complex formation (perform this step immediately before transfection):
a) Dilute 1 μg of endotoxin-free DNA in sterile PBS pH7.4 or growth media to a final concentration of 20 - 50 ng/μL and mix gently
b) Add 3 μL of Quickfectin™ to the diluted DNA and tap gently to mix well.
c) Incubate for 5 minutes at room temperature.
3. Cell culture incubation:
a) Add the mixture prepared above dropwise to the cells and gently rock the plate back-and-forth and from side-to-side to distribute the complex evenly.
b) Incubate for 24 - 72 hours before analysis of expression.
Note: With Quickfectin™, no medium change is necessary. If you wish to remove the complex, remove the medium 16 - 24 hours post-transfection and replace with fresh medium.
Protocol II: Standard protocol for transient transfection (suspension 293 cells)
1. Cell seeding:
a) On the day of transfection, seed cells at a density of 0.5 - 1 x 106 cells/ml in growth medium per well in a 12-well plate
Note: With Quickfectin™, no media change is necessary before the transfection. However, for the cells with low viability (e.g., <90%), it is recommended to collect viable cells and resuspend them gently into fresh media at transfection.
2. Complex formation (perform this step immediately before transfection)
a) Dilute 1 μg of DNA into 20-50 μL of sterile PBS pH7.4 or medium and mix gently.
b) Add 3 μL of Quickfectin™ to the diluted DNA and tap gently to mix well.
c) Incubate for 5 minutes at room temperature.
3) Cell culture incubation:
a) Add the mixture prepared above dropwise to the cells. Gently rock the plate back-and-forth and from side-to-side to distribute the complex evenly.
b) Incubate at 37°C for 24 – 72 hours before analysis of expression.
Table 1. Recommended starting transfection conditions using Quickfectin™
|
Culture Dish/Plate |
10 cm Dish |
6-well plate |
12-well plate |
24-well plate |
48-well plate |
96-well plate |
384-well plate |
|
Surface Area (cm2) |
59 |
9.6 |
3.8 |
1.9 |
0.95 |
0.32 |
0.06 |
|
Medium Volume (mL) |
10 |
2.5 |
1.0 |
0.5 |
0.25 |
0.1 |
0.03 |
|
Number of Cells Plated/Seeded |
2-5x106 |
0.5-1.0x106 |
2-5x105 |
1-2x105 |
0.5-1x105 |
20,000 - 50,000 |
5,000 - 10,000 |
|
DNA (μg) |
10-20 |
2.5-5 |
1-2 |
0.5-1 |
0.25-0.5 |
0.1-0.2 |
0.03-0.06 |
|
Quickfectin (μL) |
30-60 |
7.5-15 |
3-6 |
1.5-3 |
0.75-1.5 |
0.3-0.6 |
0.1-0.2 |
Protocol III: Standard protocol for stable transfection
Perform transient transfection as described above (using the linearized plasmid DNA that usually results in higher stable transfection efficiency if desired). 24 - 48 hours post-transfection, passage the cells (at 1:10 or higher dilution) into fresh growth medium containing selection antibiotics to generate stable pool or clones.
Protocol IV: Standard protocol for reverse transfection:
In conventional transfection, the cells are seeded before the DNA/transfection reagent complex is applied. When the plasmid DNA is applied to the plate before the cells are, such transfection is often referred to as reverse transfection. Reverse transfection is typically used in high-throughput applications when a large number of individual plasmid DNAs is arrayed onto a solid matrix, such as a glass slide or a 96-well or 384-well plate. Quickfectin™ is ideally suited for reverse transfection using above simple and quick procedure (simply in a reverse order - refer to Table 1 for more information).
The standard transfection protocols described here have been proven to result in highly efficient transfection. However, it is recommended to carefully optimize the reaction conditions for each other cell type. The following variables should be considered:
1. Cell density (%confluence at transfection for adherent cells):
The recommended confluence for most adherent cell types at transfection is 50 - 70%. For most suspension cells, Quickfectin™ can be used for transfection with a range of cell densities (0.5 - 4 x 106/ml). If needed, determine the optimal cell density for maximal efficiency.
2. Cell viability and medium compositions:
The viability for most cell types at transfection should be more than 90%. Quickfectin™ performs equally well in complete growth media (containing serum, antibiotics) and in serum-free media. Normally no medium change is necessary before or after the transfection. However, for the cells with low viability, it is recommended to collect viable cells and exchange into fresh media before or at the transfection.
3. DNA quality and quantity:
It is recommended that highly purified, endotoxin-free DNA should be used for transfection to achieve maximal efficiency. The optimal DNA amount for Quickfectin™ transfection is 1 - 3 μg per ml of cells. The DNA can be diluted with sterile PBS (pH7.4) or growth media to a concentration of 20 - 50 ng/μL before mixing with Quickfectin™.
4. Quickfectin™ to DNA ratio:
The standard ratio of Quickfectin™ to DNA is 3, i.e. 3 μL of Quickfectin™ to 1 μg of DNA for transfection of most adherent cell types. For transfection of suspensions cells, adjust Quickfectin™ from 3 - 9 μL per 1 μg of DNA transfection according to the cell density, e.g., increase the ratio to 6 for cells with a density between 1 - 2x106/mL and to 9 for a density higher than 2x106/mL to achieve the maximal efficiency.
Troubleshooting:
Low cell viability or growth
Possible cause: Cell confluence or density was not optimal (too low or high) at the time of transfection
Solution: Optimize transfection by testing various cell confluences and densities in small-scale experiments. Maintain a consistent density in future experiments to ensure reproducibility.
Possible cause: Quickfectin™ concentration or Quickfectin™:DNA ratio was too high
Solution: Reduce the amount of Quickfectin™, and optimize the ratio of Quickfectin™:DNA to minimize cytotoxicity for individual cell type.
Low transfection efficiency
Possible cause: Quickfectin™ /DNA ratio is too low or not optimized
Solution: Titrate the reagent from 3-9 μL per 1 ug of DNA transfection. Select the ratio with best transfection efficiency and the lowest toxicity for future experiments.
Possible cause: Presence of positively charged lipids or antibiotics, such as dextran sulphate or heparin, kanamycin in the media.
Solution: You may test the transfection in the absence of these components.
Product datasheet (pdf) can be downloaded here: QFT001-PDS.pdf
Additional supporting documents, including COA and MSDS are available upon request.
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