Polyethylenimine,Linear (MW-25000)

Take the 24-well plate as an example, the sample addition volume of other culture devices is referred to Table 1.

1. Prepare the cells to be transfected

1.1 Adherent cells: One day before transfection, the cells digested with trypsin, are plated (0.3-2×10⁵ cells/well), and the cell density reached 60-70% for cell transfection.

1.2 Suspended cells: On the day of transfection, before the preparation of the transfection reagent-DNA complex, cell plating is performed, and 2-5×10⁵ cells are added per 500μl growth medium.

Note: To improve transfection efficiency, use cells with good growth status and an exponential growth and survival rate of >90% for transfection.

2. Prepare the transfection agent-nucleic acid complex

2.1 Ultra-pure water is used to dissolve Polyethylenimine powder into 1mg/ml PEI Transfection Reagent and filter to remove bacteria. After the solution was prepared, please aliquot and store at 4℃ to avoid freezing and thawing.

2.2 Take 1μg plasmid DNA into a 1.5ml EP tube, add 1.25μl PEI Transfection Reagent, blow, suck, and mix, and incubate for 3min at room temperature.

Notes: (a) Normally, the dosage ratio of DNA(μg) and PEI Transfection Reagent (μl) is 1:1.25. To improve the transfection effect, adjust in the range of 1:1-1:2 to optimize the transfection effect.

(b) The optimal transfection conditions are different due to different cell types and culture conditions, and the optimal transfection ratio can be explored by pre-experiments.

2.3 Add 100μl serum-free basal medium (consistent with the culture system, no serum and no double antibody), mix gently, and incubate at room temperature for 30min to form the transfection reagent-nucleic acid complex.

Note: The transfection agent-nucleic acid complex remains stable up to 4h at room temperature.

2.4 The complexes are diluted by adding 150μl of serum-free basal medium (consistent with the culture system, serum-free without double antibody).

Table 1. The amounts of transfection reagent, DNA and culture medium per well under various culture media

Note: For cell transfection, equal magnification can be performed according to the above table, according to the actual situation.

3. Cell transfection

The original medium is removed by aspiration, and the transfection reagent-nucleic acid complex (250μl) is added to the cells in each well, gently mixed, and after incubation in the incubator for 4-16h, fresh growth medium (500μl/well) is supplemented in time.

4. Analyze transfected cells

After 36-48 hours of culture, the transfection effect can be detected by fluorescence detection, Western Blot, ELISA, RT-PCR, flow cytometry, reporter gene, or adding screening drugs to screen stable cell lines.

5. Storage conditions

Polyethylenimine powder is stored at room temperature. When prepared into solution, Polyethylenimine solution is stored at 4℃ to avoid repeated freezing and thawing.