Polyethylenimine linear (MW:10000)

This plan only provides a guide, please modify it to meet your specific needs.

1. Solution preparation

(1) Prepare storage solution: Equilibrate the cryopreserved solid to room temperature, use sterile water to dissolve Polyethylenimine linear, and prepare a storage solution with a concentration of 1 mg/mL.

Note: Unused storage solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.

(2) Prepare working solution: Before the formal experiment, dilute the storage solution with sterile water (or serum-free medium) to the required working concentration, for example: 250 μg/mL.

Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration yourself to explore. The working fluid must be prepared and used immediately.

2. Steps for transient transfection of 293T cells using Polyethylenimine linear ^[1] (from the literature, for reference only)

(1) Cell seeding: One day before transfection, 293T cells in the exponential growth phase were seeded into a 6 cm diameter culture dish. Ensure cell growth reaches 50-70% confluence.

(2 Change the culture medium: Remove the cell culture medium and replace it with serum-free culture medium.

(3) Prepare DNA-PEI complex: Prepare 240 μL of DNA solution (32 μg/mL). Add 180µL of sterile PEI solution (250 µg/mL). Vortex slightly to mix, and incubate at room temperature for 30 minutes.

(4) Add DNA-PEI complex: Add the complex dropwise to the cells and incubate in a 5% CO₂ incubator for 6 hours.

(5) Replace the culture medium: After 6hours, replace the culture medium containing the DNA-PEI complex with DMEM culture medium supplemented with fetal bovine serum (FBS).

(6) Culture and gene expression: Continue to culture in a CO₂ incubator for 48 hours, and then analyze gene expression.

Note: This experimental protocol only provides a reference for using Polyethylenimine linear in cell transfection experiments. Please modify the specific steps according to experimental needs. Please perform necessary control experiments, including untreated cells and cells using known effective transfection reagents, to evaluate transfection efficiency and cytotoxicity.

References:

[1] Wang W , Zheng Q , Yu C ,et al.WSV056 Inhibits Shrimp Nitric Oxide Synthase Activity by Downregulating[J].Frontiers in microbiology, 2021, 12:796049.