material:
Plasmid DNA
Exponentially growing eukaryotic cells
PEI (polyethyleneimine)
1×HBS (pH7.4)
formula:
PEI stock solution (100 μM): Weigh 125 mg of PEI powder and dissolve in 50 ml of 1×HBS (pH 7.4)
The 0.2 μm filter was filtered and stored at 4 ° C until use.
1×HBS (pH 7.4): Dissolve 8.76 g of NaCl in 900 ml of ultrapure water and add 20 ml of 1 M
HEPES, adjust the pH to 7.4, dilute to 1 L, filter (0.2 μm filter) and store at 4 °C for later use.
method:
1. Cell sorting: Collect cells by trypsinization, using appropriate complete medium at 4 x 105 to 8 x 105
Cell/cm2 density plated cells on 60 mm tissue culture dishes (select culture dishes according to the experiment,
The total area occupied by the cells after adherence is 70-90% of the area of ​​the culture dish. According to the condition of cell attachment
Incubate for 8-24 h in a 37 ° C incubator with 5% CO2. Transfection can begin when the cells are completely attached. Transfection
Transfer to 2 mL of pre-warmed serum-free medium before.
2. Prepare a PEI-DNA mixture: Take a 420 μL reaction volume in a 60 mm tissue culture dish as an example. quasi-
Prepare two 1.5 mL centrifuge tubes and add plasmid DNA (2-8 μg total) to 240 μL HBS in one tube
Mix and mix. In another tube, 100 μM of PEI stock solution was diluted to 10 μM with HBS.
mixing. Then add 180 μL of 10 μM PEI solution to the HBS containing DNA and mix well.
Allow to stand at room temperature for 20-30 min. Finally, add 420 μL of PEI-DNA mixture to the above single layer.
In the cell culture medium of the cells, gently shake the plate and mix and place in a 37 ° C incubator with 5% to 7% CO2.
Incubate.
Note: Before using 100 μM PEI stock solution, you need to mix it thoroughly to ensure that it is taken.
The concentration is consistent.
3. After 6-10 h of culture, replace with the serum-containing medium preheated at 37 °C, continue to culture, about 16 h
The expression of the reporter gene can be observed.
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