Reagents List
|
Product Name |
Cat.NO. |
|
40801ES |
SKOV3-DNA+siRNA co- transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Cell Washing
Aspirate the spent medium and add 3–4 mL of PBS at room temperature. Gently rock the flask for 10–20 seconds to rinse the cell monolayer, then aspirate the PBS.
- Digestion
Add 1 mL of trypsin to cover the bottom of the flask and incubate at 37°C to digest the cells.
- Detachment
Once the intercellular spaces widen and the cells become rounded, gently pipette the trypsin over the surface to dislodge the cells.
- Termination
Add 2–3 mL of complete medium to stop the digestion. Pipette the mixture up and down to ensure a homogeneous cell suspension.
- Seeding
Collect the suspension and centrifuge at 900 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in fresh medium, distribute into new flasks, and add sufficient medium to continue culture.
Cell Transfection (6-Well Plate)
I. Preparation
1. Cell Preparation: 24 hours before transfection, cells were detached with trypsin, resuspended, and counted. They were then seeded into 6-well plates (or other culture vessels) at an appropriate density, with 2 mL of complete growth medium added per well. The goal is to achieve 60–80% confluency at the time of transfection.
2. Reagent Preparation:
- siRNA Preparation: The siRNA was synthesized and diluted to a concentration of 100 μM.
- Transfection Reagent: Allow Hieff Trans™ Booster Transfection Reagent to warm to room temperature. Gently mix before use.
- Serum-Free Medium: Prepare serum- and antibiotic-free basal medium (e.g., Opti-MEM) for diluting DNA and transfection reagent.
II. Complex Formation
1. Dilute DNA: In a sterile microcentrifuge tube, add 125 μL of serum-free medium, 1 μg of plasmid DNA, and 2 μL of Enhancer. Mix gently by pipetting to obtain the diluted DNA solution.
2. Dilution of siRNA
In a sterile microcentrifuge tube, add 125 μL of serum-free medium and 1 μL of siRNA (100 μM stock solution). Mix gently by pipetting to obtain the diluted siRNA solution.
3. Dilute Transfection Reagent: In a separate sterile microcentrifuge tube, add 125 μL of serum-free medium and 7.5 μL of Booster transfection reagent. Mix gently by pipetting (prepare two aliquots).
4. Combine and Incubate: Add the diluted DNA-siRNA solution to the diluted transfection reagent tube. Mix gently by pipetting. Incubate at room temperature for 10–15 minutes to form DNA-transfection reagent complexes.
III. Transfection
1. Medium Replacement: Prior to transfection, carefully aspirate the original medium and replace it with 2 mL of fresh, pre-warmed complete medium.
2. Add Complexes: Add the incubated DNA/siRNA-transfection reagent complexes dropwise and evenly to the cells. Gently rock the plate to distribute.
3. Incubation: Return cells to a 37°C, 5% CO₂ incubator for culture.
IV. Post-Transfection Handling
1. Medium Replacement: Observe cell morphology 4–6 hours post-transfection. If significant cytotoxicity is observed, aspirate a portion of the supernatant and replenish it with 2 mL fresh, pre-warmed complete medium. If cells appear healthy, medium replacement is not necessary. In this experiment, the medium was changed 8 hours after transfection.
2. Analysis: After 24–72 hours, assess transfection efficiency or functional outcomes based on experimental design (e.g., fluorescence observation, mRNA or protein detection). In this experiment, proteins were extracted and analyzed 72 hours after transfection.
Tips:
1. If significant cytotoxicity occurs, replace medium after 6 h or reduce Enhancer volume by half to mitigate toxicity.
2. If transfection efficiency is low, increasing the amount of transfection reagent may improve results.
3. For optimal performance, dilute DNA and transfection reagent in Opti-MEM rather than DMEM.
4. The transfection system is compatible with serum and antibiotics; however, DNA and reagent dilutions must be performed in serum- and antibiotic-free medium.
Experimental Results Analysis
SKOV3 human ovarian cancer cells were transfected with DNA and siRNA using Yeasen Booster, and the transfection efficiency was evaluated by Western blot.
Figure 1. SKOV3 human ovarian cancer cells were transfected with DNA and siRNA using Yeasen Booster transfection reagent. The results demonstrated that Yeasen Booster 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 |
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