Cell transfection method

Transfection is a specialized technique for introducing exogenous genes into cells. With the deepening of research on gene and protein function, transfection has become a basic method often involved in laboratory work. Transfection can be broadly classified into three types: physical mediation, chemical mediation, and biological mediation. Electroporation, microinjection, and gene guns are examples of physical introduction of genes into cells; many chemically mediated methods, such as classical calcium phosphate coprecipitation, lipofection, and multiple cationic species The technology; biologically mediated methods, with relatively primitive protoplast transfection, and nowadays more common viral-mediated transfection techniques.

The ideal cell transfection method should have the advantages of high transfection efficiency and small cytotoxicity. Virus-mediated transfection technology is currently the most efficient method of transfection, and has the advantage of low cytotoxicity. However, the preparation procedure of the virus transfection method is complicated, and it is often highly selective to cell types, and it is difficult to popularize in a general laboratory. Other physical and chemically mediated transfection methods have their own characteristics.

It should be pointed out that no matter which transfection technique is used, it may be necessary to optimize the transfection conditions in order to obtain optimal transfection results. There are many factors that affect transfection efficiency, from cell type, cell culture conditions and cell growth status, to operational details of transfection methods.

First, cell passage

1. Test preparation: 200ul/1ml Tip head each box (all items need to be autoclaved), alcohol cotton ball, waste liquid tank, test tube rack, micro pipette, marker pen, culture dish, centrifuge tube.

2. Discard the medium in the Petri dish and wash twice with 1 ml of PBS solution.

3. Add 1 ml of Trypsin solution to the Tip head and digest for 1 minute (37 ° C, 5% CO 2 ). The wall of the culture flask was patted by hand and the cells were observed to completely fall off the wall.

4. Stop the reaction by adding 1 ml of serum-containing medium.

5. Use the Tip head to breathe multiple times to completely disperse the cells.

6. Place the culture solution in a centrifuge tube and centrifuge at 1000 rpm for 5 min.

7. Resuspend the cells with the culture medium. After the cells are counted, select 0.8X106 cells and add a 35mm culture dish.

8. Add the appropriate volume of complete culture to the centrifuge tube, mix the cells and gently add to the culture dish to distribute them evenly.

9. Transfer the culture dish to a CO2 incubator and transfect the next day.

Second, cell transfection

1. Preparation of transfection reagents

1 Add 400 ul of denuclease water to the tube and shake for 10 seconds to dissolve the lipid.

2 After shaking, store the reagent at -20 °C and shake it before use.

2. Select the appropriate mix ratio (1:1-1:2/liposome volume: DNA mass) to transfect the cells. Add a suitable volume of serum-free medium to a transfection tube. Add DNA of the appropriate mass of MyoD or EGFP, shake it and then shake it again by adding the appropriate volume of transfection reagent.

3. Leave the mixture at room temperature for 10-15 minutes.

4. Aspirate the medium from the plate and wash once with PBS or serum-free medium.

5. Add the mixture and place the cells back in the incubator for one hour.

6. After the time, decide whether to remove the mixture according to the cell type, then add the complete medium and continue to culture for 24-48 hours.

Third, the second cell passage

1. Observe the results 24 hours after transfection and record the expression of green fluorescent protein.

2. Perform cell passage again and re-transfer the cells into the culture dish according to the appropriate density of 0.8X105 cells/35mm culture dish.

3. After 24 hours of incubation under normal conditions, fix it according to the staining requirements.

For more parameters and products, please refer to the website of Shanghai Shujun Instrument Equipment Co., Ltd.,

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