1. 1-3 × 10⁵ cells / well were inoculated in a 6-well plate, 2ml of complete medium was added, and cultured overnight in a CO₂ incubator at 37 ℃.

2. When the cells grow to 50-80% monolayer, prepare the following solution in the sterile centrifuge tube:
(1) Solution A:    Dilute 1-2 μg of ultrapure DNA to be transfected into 100 μl serum-free medium.
(2) Solution B:    Dilute 2-25 μl LipofectAMINE to 100 μl serum-free medium.

3. Mix solution a and B, mix gently, and place at room temperature for 15-45min.

4. The cells were washed with 2ml serum-free medium, 0.8ml serum-free medium / pore was added, the liposome complex was dropped into the pore, gently shaken and mixed, and incubated in CO₂ incubator at 37 ℃ for 2-24hr.

5.The transfection medium was replaced with complete medium and the culture continued.

6.After 24-72 hr, the expression or passage of the protein was detected and the stable expression strains were screened by adding selective antibiotics.