Reagents List
|
Product Name |
Cat.NO. |
|
Hieff Trans™ Booster DNA/RNA Transfection Reagent (Lipo3000 alternative) |
40801ES |
COLO320DM siRNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Washing: Aspirate the old medium, add 3–4 mL of room-temperature PBS, gently swirl to rinse for 10–20 seconds, then aspirate.
- Digestion: Add 1 mL of trypsin to cover the bottom of the flask and incubate at 37°C for digestion.
- Detachment: When cells begin to separate and round up, gently pipette the trypsin up and down to dislodge the cells.
- Termination: Add 2–3 mL of complete medium to inactivate the trypsin, then pipette to resuspend and create a uniform cell suspension.
- Seeding: Transfer the suspension to a centrifuge tube, centrifuge at 200 × g for 3–5 minutes, aspirate the supernatant, resuspend the cell pellet in fresh medium, seed into new culture vessels, and add fresh medium to the required volume for continued culture.
Cell Transfection (6-Well Plate)
I. Preparation
1. Cell Preparation: 24 hours before transfection, cells were trypsinized, resuspended, and counted. Cells were then seeded into 6-well plates (or other vessels) with 2 mL of complete medium per well at an appropriate density to ensure 60–80% confluency at the time of transfection.
2. Reagent Preparation:
- siRNA: Synthesize high-quality siRNAs with the initial concentration adjusted to 20 μM.
- Transfection Reagent: Allow Hieff Trans™ RNAiBoost Transfection Reagent to equilibrate to room temperature before use. Mix gently by pipetting.
- Serum-free Medium: Prepare a serum-free base medium (e.g., Opti-MEM) for diluting siRNA and transfection reagent.
II. Cell Transfection Protocol
1. Dilute siRNA: Add 125 μL of serum-free medium and 5 μL of siRNA (final concentration 50 nM) to a sterile centrifuge tube. Mix by pipetting.
2. Dilute Transfection Reagent: Dilute transfection reagent: Add 125 μL of serum-free medium and 7.5 μL of siRNA transfection reagent to a sterile centrifuge tube. Mix by pipetting.
3. Mix and incubate: Add the diluted siRNA solution to the tube containing the diluted transfection reagent. Mix gently by pipetting. Incubate at room temperature for 10–15 minutes to allow the formation of stable RNA-transfection reagent complexes.
III. Cell Transfection
1. Medium Replacement: Prior to transfection, carefully aspirate the original culture medium and replace it with 2 mL of fresh, pre-warmed complete medium.
2. Add Complexes: Add the incubated RNA-transfection reagent complexes dropwise to the cell culture medium. Gently rock the culture plate to ensure thorough mixing.
3. Incubation: Return the cells to a humidified incubator at 37°C with 5% CO₂ for culture.
IV. Post-Transfection Handling
1. Medium Replacement: 4–6 hours post-transfection, check cell morphology. If significant toxicity is observed, aspirate the medium containing complexes and replace with 2 mL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional.
2. Analysis: Typically, RNA is extracted for analysis 48 hours after transfection.
Tips:
1. If significant cytotoxicity is observed after transfection, consider replacing the medium at 6 hours or halving the reagent dosage to avoid cytotoxicity issues.
2. If the transfection efficiency is relatively low, increasing the amount of transfection reagent can effectively improve it.
3. Opti-MEM is recommended for diluting siRNA and transfection reagents.
4. The transfection reagent is compatible with serum and antibiotics; however, the medium used to dilute the plasmid and transfection reagent must be serum-free and antibiotic-free.
5. Strictly adhere to the recommended dosages in the table for preparing the transfection complex. If you need to increase or decrease the dosage, do so proportionally. For example, for a 6-well plate, if the final concentration is 50 nM, prepare 100 µL of transfection complex according to the manual. If you need to double the dosage, prepare 200 µL of transfection complex and add it to the well. Conversely, if you halve the dosage, simply add 50 µL of the prepared 100 µL transfection complex to the well.
Experimental Results Analysis
COLO320DM human colon cancer cells were transfected with UHRF1 siRNA using Yeasen Booster, and the transfection efficiency was evaluated by Western blot.
Figure 1. COLO320DM human colon cancer cells were transfected with UHRF1 siRNA using Yeasen Booster transfection reagent. The results demonstrated that Yeasen LipoBooster 3000 is capable of highly efficient transfection.
Different Cell Culture Vessel Transfection Volumes (for reference only):
|
Culture vessel |
Medium Volume |
DNA Transfection |
siRNA Transfection (Final Concentration 50 nM) |
||||
|
Volume of Medium |
Volume of Opti-MEM Complex |
DNA(μg) |
Booster Transfection Reagent (μL) |
Transfection Enhancer (μL) |
Volume of siRNA (Initial Concentration 20 μM) |
Booster Transfection Reagent (μL) |
|
|
96-well |
100 μL |
2×5 μL |
0.1 |
0.2 |
0.2 |
0.25 μL |
0.3 |
|
48-well |
250 μL |
2×12.5 μL |
0.25 |
0.5 |
0.5 |
0.625 μL |
0.75 |
|
24-well |
500 μL |
2×25 μL |
0.5 |
1 |
1 |
1.25 μL |
1.5 |
|
12-well |
1 mL |
2×50 μL |
1 |
2 |
2 |
2.5 μL |
3 |
|
6-well |
2 mL |
2×125 μL |
2.5 |
5 |
5 |
5 μL |
7.5 |
|
60 mm |
5 mL |
2×250 μL |
5-10 |
10-20 |
10-20 |
12.5 μL |
20 |
|
10 cm |
10 mL |
2×500 μL |
15-25 |
30-50 |
30-50 |
25 μL |
40 |
|
T25 |
6 mL |
2×250 μL |
6-12 |
12-24 |
12-24 |
15 μL |
24 |
|
T75 |
15 mL |
2×750 μL |
20-40 |
40-80 |
40-80 |
37.5 μL |
60 |
[Note]: The volumes provided in this table are for reference only and are suitable for both suspension and adherent cells. For suspension cells, the test can be performed at a cell density of 0.5–1×10⁶ cells/mL. The actual amounts of DNA and Booster DNA/RNA transfection reagent should be optimized depending on cell type and other experimental conditions. It is recommended to maintain a ratio between 1:0.5 and 1:5 (DNA : Booster transfection reagent). The amount and experimental conditions for mRNA are the same as those for DNA. If the cells are particularly fragile and excessive cell death is observed, better results may be achieved by reducing the amount of enhancer by half.
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