PEI Transfection Reagent

Using Method

Using a 24-well plate as an example, the sample volume for other culture devices is referred to in Table 1.

1.Prepare the cells to be transfected

1.1 Adherent cells: The day before transfection, seed the cells that have been digested with trypsin (0.3-2 x 10⁵ cells/well) until the cell density reaches 60-70% for transfection. 1.2 Suspension cells: On the day of transfection,  before preparing the transfection reagent-DNA complex, first seed the cells. Add 2-5 x 10⁵ cells to every 500 μL of growth medium.

1.2 Suspension cells: On the day of transfection, before preparing the transfection reagent-DNA complex, first seed the cells. Add 2-5 x 105 cells to every 500 μL of growth medium. Note: To improve transfection efficiency, it is recommended to use cells in good growth condition, in the exponential phase, and with a survival rate >90% for transfection.

2.Prepare the transfection reagent-nucleic acid complex

2.1 Transfer 1 μg of plasmid DNA into a 1.5 mL EP tube, add 1.25 μL of PEI Transfection Reagent, and mix well by pipetting. Incubate at room temperature for 3 minutes. Note: a) Typically, the ratio of DNA (μg) to PEI Transfection

Reagent (μL) is 1:1.25. To enhance transfection results, adjust the ratio within the range of 1:1 to 1:2 to optimize the transfection effect. b) The optimal transfection conditions vary depending on the cell type and culture conditions. It's advisable to conduct preliminary experiments to determine the best transfection ratio.

2.2 Add 100 μL of serum-free basic culture medium (consistent with the culture system, without serum and without double antibody) and mix gently. Incubate at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex. Note: This transfection reagent-nucleic acid complex remains stable at room temperature for up to 4 hours. 2.3 Add 150 μL of serum-free basic culture medium (consistent with the culture system, without serum and without double antibody) to dilute the complex.

Table 1. Amount of transfection reagent, DNA, and culture medium per well in various culture media

Note: For cell transfection, you can refer to the above table and scale up proportionally based on actual conditions.

3.Cell Transfection

Remove the original culture medium and add the transfection reagent-nucleic acid complex (250 μL) to each well containing cells. Mix gently and incubate in the culture incubator for 4-16 hours. Afterward, promptly supplement with fresh growth medium (500 μL/well).

4.Analyze Transfected Cells

After culturing the transfected cells for 36-48 hours, you can use fluorescence detection, Western Blot, ELISA, RT-

PCR, flow cytometry, reporter gene assays, etc., to check the transfection efficiency. Alternatively, you can also add screening drugs for the selection of stable cell lines.

Storage Conditions

Store at 4°C for 12 months.

Avoid repeated freeze-thaw cycles.