Description
Transfection reagent for hard-to-transfect cells
GenieFect 3000 Transfection Reagent is a next-generation lipid transfection reagent that delivers plasmid DNA and other nucleic acids with high efficiency, including into sensitive and hard-to-transfect cell lines. It is optimised for strong expression with minimal cytotoxicity.
The kit combines GenieFect 3000 Reagent, which forms liposome/nucleic acid complexes and rapidly releases the nucleic acid inside cells, with GenieFect 3000 Enhancer Reagent, which helps DNA enter the nucleus quickly. Together they give higher efficiency with large plasmids and difficult cells. One reagent handles plasmid DNA, mRNA, siRNA and co-transfection in adherent or suspension cells. Serum and antibiotics do not affect performance, so complexes can be added directly to complete medium. Suitable cells include HCT116, Raw264.7, A549, Jurkat, HeLa, MDCK, NIH3T3, MDA-MB-231, Vero, H9C2, C2C12, COS7 and Huh7. For research use only.
Key Features
| Superior performance | Efficiently transfects large plasmids and difficult-to-transfect cells. | |
| Broad compatibility | One reagent for plasmid DNA, mRNA, siRNA and co-transfection. | |
| Maintains cell viability | Gentle on cells with low toxicity. | |
| Serum-compatible | Complexes can be added directly to complete medium containing serum and antibiotics. |
Specifications
| Method | Liposome-mediated transfection with nuclear-entry enhancer |
| Nucleic acids | Plasmid DNA, mRNA, siRNA; co-transfection |
| Cell types | Most eukaryotic adherent and suspension cells |
| Medium | Compatible with serum and antibiotics |
| Shelf life | 2 years |
Components
| Components | Amount |
| GenieFect 3000 Reagent | 1.5 ml / 3 × 1.5 ml |
| GenieFect 3000 Enhancer Reagent | 1.5 ml / 3 × 1.5 ml |
Storage
Store at 2 ~ 8°C.
Transient transfection in a 24-well plate is given as an example.
- Seed the cells
- About 24 h before transfection, seed cells at about 0.5–2 × 105 cells/well.
- Transfect when cells reach 70%–90% confluence.
- Form the transfection complexes
- In a 1.5 ml sterile tube, mix 25 μl Opti-MEM medium with 1.5 μl GenieFect 3000 Reagent by gentle pipetting.
- In a second 1.5 ml sterile tube, mix 25 μl Opti-MEM medium with 0.5 μg DNA by gentle pipetting. Add 1 μl GenieFect 3000 Enhancer Reagent and mix gently again.
- Add the DNA/Enhancer/Opti-MEM mixture to the GenieFect 3000 Reagent/Opti-MEM mixture and pipette gently. Leave at room temperature for 10 min.
- Transfect
- Add the complexes to the culture medium and gently rock the plate to distribute them evenly.
- If needed, replace with fresh serum-containing medium before transfection to prevent cell death from high density or nutrient depletion.
- Incubate for 24–48 h. If the medium must be changed after transfection, do so 6–12 h after adding the complexes.
- Harvest
- Harvest cells for downstream experiments.
Recommended starting conditions for different culture formats
| Culture format | Complete growth medium | Serum-free medium | GenieFect 3000 Reagent (DNA) | Enhancer Reagent | Plasmid | GenieFect 3000 Reagent (siRNA) | siRNA |
| 96-well plate | 100 μl | 10 μl | 0.15 μl, 0.3 μl | 0.2 μl | 0.1 μg | 0.3 μl | 3 pmol |
| 24-well plate | 500 μl | 50 μl | 0.75 μl, 1.5 μl | 1.0 μl | 0.5 μg | 1.5 μl | 15 pmol |
| 6-well plate | 2.0 ml | 250 μl | 3.75 μl, 7.5 μl | 5.0 μl | 2.5 μg | 7.5 μl | 75 pmol |
| 6 cm dish | 5.0 ml | 500 μl | 8.25 μl, 16.5 μl | 11–22 μl | 5.5–11 μg | 17 μl | 166 pmol |
| 10 cm dish | 10.0 ml | 1.0 ml | 21.7 μl, 43.4 μl | 28–56 μl | 14–28 μg | 43 μl | 434 pmol |
- For siRNA or mRNA, follow the DNA protocol but do not add Enhancer Reagent when diluting the RNA.
- These conditions are a starting point; optimise for your cells.
Q1: Can I transfect siRNA or mRNA with this reagent?
- Yes. Follow the DNA protocol, but do not add the Enhancer Reagent when diluting siRNA or mRNA. Recommended siRNA amounts for each culture format are given in the protocol table.
Q2: Do I need to remove serum or antibiotics before transfection?
- No. Serum and antibiotics do not affect transfection, so complexes can be added directly to complete medium. However, dilute the nucleic acid and reagent in serum-free medium when forming complexes.
Q3: When can I change the medium after transfection?
- If a medium change is needed, do it 6–12 h after adding the transfection complexes.
Q4: How can I improve transfection efficiency?
- Transfect at 70%–90% confluence, keep the seeding ratio consistent, use high-purity, sterile, endotoxin-free DNA, use the Enhancer Reagent for DNA, and optimise the reagent amounts for your cell type.
