Reagents List
|
Product Name |
Cat.NO. |
|
40801ES |
Jurkat-mRNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
- Cell Counting
Remove the cells from the incubator, mix the cell suspension thoroughly by pipetting, and use a cell counter to detect cell viability and density.
- Cell Passaging (Direct Dilution Method)
Based on the counting results, passage the cells by direct dilution to a density of approximately 5 × 10⁵ cells/mL.
- Cell Passaging (Half-Medium Change Method)
If the supernatant of the cells to be passaged appears yellowish, after removing the cells from the incubator, allow them to remain undisturbed. Gently tilt the culture plate to let the cells settle at the bottom, then use a pipette to aspirate the supernatant. Subsequently, add fresh complete medium, and proceed with cell counting and passaging.
- Cell Passaging (Centrifugation-Medium Change Method)
If the cell condition is poor, passaging via low-speed centrifugation can be performed. Transfer the cell suspension to be passaged into a sterile centrifuge tube, centrifuge at 200 ×g for 3–5 minutes, resuspend the cells in fresh complete medium, and then proceed with cell counting and passaging.
Cell Transfection (24-well-plate)
I. Preparation
1. Cell Preparation:
Prior to transfection, centrifuge the cells at 800 rpm, collect the cell suspension, and perform cell counting (cell viability can also be assessed; a viability of >95% is recommended). Seed the cells into 24-well plates at a density of 1 × 10⁶ cells/mL, adding 500 μL of complete medium per well (referring to the total medium volume).
2. Reagent Preparation:
- mRNA: Use high-quality mRNA.
- 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 mRNA:
In a sterile microcentrifuge tube, add 25 μL of serum-free culture medium, followed by 1 μg mRNA in the transfection mix. Gently pipette to mix thoroughly.
2. Dilute Transfection Reagent:
In a separate sterile tube, combine 25 μL of serum-free culture medium with 3 μL of Booster Transfection Reagent. Mix gently by pipetting.
3. Complex Formation:
Add the diluted mRNA 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 mRNA–transfection reagent complexes.
III. Transfection
1. Medium Replacement: Before adding the complexes, carefully remove the old culture medium and replace it with 2 mL fresh, pre-warmed complete medium.
2. Add Complexes: Add the incubated saRNA-transfection reagent complexes (total volume approx. 250 μL) 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: 4–6 hours post-transfection, carefully examine cell morphology. If significant cytotoxicity is observed, aspirate the medium containing the complexes and replace with 2 mL fresh, pre-warmed complete medium. If cells appear healthy, medium change is not required.
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). In this experiment, fluorescence microscopy was used to observe the cells 20 hours post-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 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
GFP-mRNA was transfected into Jurkat cell using Yeasen Booster, and transfection efficiency was analyzed by fluorescence microscopy.
Figure 1. mRNA transfection in Jurkat cell using Yeasen Booster, the results demonstrated that Yeasen Booster facilitated 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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