Reagents List

Product Name	Cat.NO.
Hieff Trans™ Booster DNA/RNA Transfection Reagent	40801ES

HePG2-DNA 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 into a 6-well plate (or other culture vessels) at an appropriate density with 2 mL of complete medium per well. The goal is to achieve 60–80% confluency at the time of transfection.

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 125 μL serum-free medium, followed by 2.5 μg of plasmid DNA. Mix gently. Then add 5 μL of Enhancer, and mix thoroughly by pipetting to obtain the diluted DNA solution.

2. Dilute Transfection Reagent: In another sterile tube, add 125 μL serum-free medium and 5 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 Replacement: Before transfection, carefully aspirate the old medium from the cells and replace with 2 mL fresh, pre-warmed complete medium.

2. Add Complexes: Add the incubated DNA-transfection reagent complexes (approximately 250 μL) dropwise and evenly into the wells. Gently shake the plate to ensure even distribution.

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: 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

Plasmid DNA (6 kbp) was transfected into HePG2 cells using Yeasen Booster DNA/RNA Transfection Reagent versus L*3000, and transfection efficiency was assessed by fluorescence microscopy.

 Yeasen-Booster                       L*3000

Figure 1. Plasmid DNA transfection in HePG2 cell using Yeasen Booster DNA/RNA Transfection Reagent versus L*3000. The result demonstrates superior DNA transfection efficiency of Booster.

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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