PEI transfection method

PEI transfection method

Formula:
PEI stock solution (100 μM): Weigh 125 mg of PEI powder, dissolve it in 50 ml of 1×HBS (pH 7.4), filter through a 0.2 μm filter, and store at 4°C for later use.
1×HBS (pH 7.4): Dissolve 8.76 g NaCl in 900 ml ultrapure water, add 20 ml 1 M HEPES, adjust the pH to 7.4, dilute to 1 L, filter (0.2 μm membrane) and store Reserve at 4°C.
Method:
1. Cell plate: Harvest cells by trypsinization and plate cells on a 60 mm tissue petri dish at a density of 4 × 105 to 8 × 105 cells/cm2 with appropriate complete medium (choose the petri dish according to the experimental needs, so that the cells The total area occupied by the adherent reaches 70-90% of the area of ​​the petri dish). Incubate for 8-24 h in a 37°C incubator with 5% CO2 depending on the adherence of cells. Transfection can be started when cells adhere completely. Change into 2 mL of pre-warmed serum-free medium before transfection.Plastic Petri Dish,Petri Dish 60mm,Sterile Petri Dish
2. Prepare the PEI-DNA mixture: Take a 60 mm tissue petri dish with a total reaction volume of 420 μL as an example. Prepare two 1.5 mL centrifuge tubes, add plasmid DNA (preferably total 2-8 μg) into 240 μL HBS, and mix well. In another tube, dilute the 100 μM PEI stock solution to 10 μM with HBS and mix well. Then add 180 μL of 10 μM PEI solution to HBS containing DNA, mix well and let stand at room temperature for 20-30 min. Finally, the 420 μL PEI-DNA mixture was added dropwise to the cell culture medium of the above monolayer cells, the culture dish was shaken gently to mix, and then incubated in a 37°C incubator containing 5% to 7% CO2.Plastic Petri Dish,Petri Dish 60mm,Sterile Petri Dish
Note: Before each use of 100 μM PEI stock solution, it needs to be thoroughly mixed to ensure the same concentration.
3. After culturing for 6-10 hours, replace with serum-containing medium pre-warmed at 37°C and continue to culture. The expression of the reporter gene can be observed in about 16 hours.