Abstract: This experiment describes several methods for lipofection.
Experimental Principles <br> The liposome (LR) reagent is a mixture of cationic liposomes DOTMA and DOPE (1:1). It is suitable for transfecting DNA into suspension or adherent culture cells and is one of the most transfection methods under current conditions. The transfection rate is higher than that of the calcium phosphate method, which is 5-100 times higher than that, and can transfect DNA and RNA into various cells. When transfecting with LR, it is first necessary to optimize the transfection conditions, and the amount and time of action of the batch of LR for transfecting a particular cell should be found, and it is necessary for each batch of LR. First fix the amount of DNA and the time that the DNA/LR mixture interacts with the cells. DNA can be started from 1-5ug and incubation time 6h. According to these two parameters, the corresponding LR requirement curve is drawn, then LR and The optimal dose of both DNA determines the time of transfection. Because LR has certain toxicity to cells, the transfection time should not exceed 24h.
Cell types: Any of various cells such as COS-7, BHK, NIH-3T3, Hela, and Jurkat can be used as recipient cells.
Experimental procedure
1. Operation steps (method 1):
1) A 6-well culture plate was taken, and 2 ml of a medium containing (1-2) x 10 5 cells was added to each well at 37 ° C, 18% CO 2 medium at 40%-60% confluence.
2) Preparation of transfection solution: The following two solutions (the amount used for transfection of one empty cell) were prepared in a polystyrene tube.
Solution A: Dilute the DNA in serum-free medium to a concentration of 1-10 ug, with a final dose of 100 ul.
Solution B: Dilute LR with serum-free medium to a final concentration of 2-50 ug, with a final dose of 100 ul.
Gently mix the A and B solutions and set them at room temperature for 10-15 minutes. Later, microturbidity will appear, but it does not hinder transfection.
3) Transfection preparation: rinse 2 times with 2 ml serum-free medium, and add 1 ml serum-free medium.
4) Transfection: The A/B complex was slowly added to the medium, shaken, placed in a 37 ° C incubator for 6-24 h, aspirate the serum-free transfection solution, and exchanged for normal medium to continue the culture.
5) Other treatments: such as observation, screening, testing, etc. are the same as other transfection methods.
6) Note: Do not add serum during transfection, serum has a great impact on transfection efficiency.
2. Rapid lipofection method (Method 2):
1) Cells were seeded at 5 x 105 cells/well in 6-well plates for 24 h to achieve a 50%-60% plate bottom area.
2) Prepare a DNA-liposome complex in a test tube.
a. Dilute PSV1-neo plasmid DNA or donor DNA in 1 ml serum-free DMEM.
b. Rotate for 1 s, then add the liposome suspension and rotate.
c. Leave at room temperature for 5-10 min to allow DNA to bind to the liposomes.
3) Discard the old solution in the cells, wash the cells once with 1 ml of serum-free DMEM, discard them, and add 1 ml of the DNA-liposome complex directly to each well, and incubate at 37 ° C for 3-5 days.
4) DMEM containing 20% ​​fetal bovine serum was added to each well and culture was continued for 14-24 hours.
5) Aspirate the DMEM-DNA-liposome mixture into fresh DMEM containing 10% fetal calf serum, 2 ml/well, and culture for another 24-48 h.
6) Collect cells by cell scraping or digestion for analysis and identification.
3. Stable lipofection method:
1) Inoculate the cells as described above, and the cells can be used for transfection up to 50% of the plate bottom area.
2) Preparation of transfected cells by DNA-liposome complexes.
3) 1 ml of DMEM containing 20% ​​fetal bovine serum was added to each well, and cultured at 37 ° C for 48 hours.
4) Aspirate DMEM, dilute the cells with G418 selective medium, and allow the cells to grow for a certain period of time, and screen the transfected clones by the cell clone screening method.
4. Invitrogen's Lipofectamine 2000 transfects monolayer adherent cells in 24-well plates. (For other methods, please refer to the manufacturer's protocol)
1) One day before transfection, 0.5-2×105 cells were seeded in a 24-well culture plate, and 500 ul of complete medium containing no antibiotics was added to ensure that the cells confluence reached 90-95% during transfection.
2) Prepare the complex a. Dilute 0.8ug of DNA into 50ul of serum-free antibiotic-free medium and mix gently.
b. Dilute 2ul Lipofectamine2000 in 50ul serum-free antibiotic-free medium, gently rub and incubate for 5 minutes at room temperature. Note: Must be done within 25 minutes.
c. Mix them after 5 minutes, gently knead and incubate for 20 minutes at room temperature.
3) Aspirate the culture medium and wash the cells twice with PBS or serum-free medium (preferably).
4) Add the complex (total volume 100 ul) to the culture well, and shake the culture plate back and forth to make it evenly distributed.
5) After incubating the cells in the incubator for 4-6 hours, the serum-containing medium can be replaced to remove the complex (or not).
6) After 24 to 48 hours, the gene expression of the transferred gene can be observed.
7) Stable transfection: cells were passaged at 1:10 (or higher) after 24 h of serum-containing medium, and screened for 1 day later.
8) Optimization: to ensure that the cell confluence rate is 90-95% (higher); the ratio of DNA/Lipofectamine2000 is 1:0.5～1:5, and the average cell is 1:2～3.
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