Reagents List
|
Product Name |
Cat.NO. |
|
40801ES |
K562-DNA 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 (6 cm Dish)
I. Preparation
1. Cell Preparation: Prior to transfection, centrifuge at 800 rpm, collect the cell suspension, and perform cell counting. (Cell viability testing is also recommended; viability should be >95%).
2. Reagent Preparation:
- Plasmid DNA: Use high-purity plasmid DNA (e.g., purified by kit), ideally at a concentration >1000 ng/μL.
- 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 250 μL serum-free medium followed by 10 μg plasmid DNA. Mix gently. Then add 20 μL Enhancer, and mix again to obtain diluted DNA.
2. Dilute Transfection Reagent: In another sterile tube, add 250 μL serum-free medium and 20 μL Hieff Trans™ Booster Transfection Reagent. Mix gently.
3. Combine and Incubate: Add the diluted DNA 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 Change: Before transfection, carefully aspirate the old medium and replace with 2 mL pre-warmed complete medium.
2. Add Complexes: Add the incubated DNA-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: 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: After 24–72 hours, assess transfection efficiency or functional outcomes based on experimental design (e.g., fluorescence observation, mRNA or protein 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 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
K562 human chronic myeloid leukemia cells were transfected with plasmid DNA (8 kb) using Yeasen Booster, and the transfection efficiency was evaluated by flow cytometry.
Figure 1. K562 human chronic myeloid leukemia cells were transfected with plasmid DNA 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 |
Try it for free!
Scan the QR code below to submit your shipping information and receive Hieff Trans™ Booster transfection reagent free of charge.
Get a free sample of Hieff Trans™ Booster and validate its performance in your own lab.
👉 [Request Sample Now]. Worldwide Shipping.
