Reagents List
|
Product Name |
Cat.NO. |
|
40801ES |
MDA-MB-231 sgRNA 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, detach cells using trypsin, resuspend, and perform cell counting.
Seed cells at an appropriate density into a 6-well plate with 2 mL complete growth medium per well/flask, respectively.
- The target confluency at the time of transfection should be 60%–80%.
2. Reagent Preparation:
- sgRNA: Prepare the sgRNA required for the experiment in advance.
- Transfection Reagent: Allow Hieff Trans™Booster 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 saRNA and transfection reagent.
II. Preparation of Transfection Complexes
1. Dilute sgRNA:
In a sterile microcentrifuge tube, add 125 μL of serum-free medium and 5 μL of sgRNA. Mix gently by pipetting to obtain the diluted siRNA solution.
2. Dilute Transfection Reagent:
In a separate sterile tube, combine 125 μL of serum-free culture medium with 5 μL of Booster Transfection Reagent. Mix gently by pipetting.
3. Complex Formation:
Add the diluted sgRNA solution to the diluted Booster solution. Mix gently by pipetting. Incubate the mixture at room temperature for 10–15 minutes to allow formation of stable saRNA–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 siRNA-transfection reagent complexes dropwise and evenly to the cell culture medium, and gently rock the culture plate to mix.
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 the medium containing complexes and replace it with 2 mL of pre-warmed complete medium. If cells appear healthy, medium replacement is optional; however, in this experiment, the medium was changed to complete medium at 6 hours post-transfection.
2. Analysis: Incubate for an additional 24–72 hours, then assess transfection efficiency or function according to the experimental design (e.g., fluorescent protein expression, mRNA or protein level detection).
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 saRNA and transfection reagent in Opti-MEM rather than DMEM.
4. The transfection system is compatible with serum and antibiotics; however, saRNA and reagent dilutions must be performed in serum- and antibiotic-free medium.
Experimental Results Analysis
MDA-MB-231 human breast cancer cells were transfected with sgRNA using Yeasen Booster, and the transfection efficiency was evaluated by Western blot.
Figure 1. MDA-MB-231 human breast cancer cells were transfected with sgRNA 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 |
[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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