Cell transfection
Cell transfection is to introduce foreign molecules such as DNA, u0026 u200b u200bNBSP; RNA, such as DNA, u0026 u200b u200bNBSP; RNA, such as DNA, u0026 u200b u200bNBSP; RNA. With the continuous development of molecular biology and cell biology research, transfection has become a conventional tool for studying and controlling eukaryotic cellular function. It is increasingly widely used in research genetic functions, regulatory gene expression, mutation analysis, and protein production.
Because different cells differ in physiological metabolism and split, the researchers will need to choose from the exogenous genetic material guides, in the process of entering the cells
. At present, the method of introducing foreign substance into cell is mainly divided into three categories:
1, liposome (or liposome alternative) transfection;
2, electric rotation;
3, virus Infect.
These three methods have the advantages and disadvantages. Liposomes or liposome alternative transfections are simple, low price, but the cytotoxicity is large. Moreover, there is a very low efficiency of liposome by liposome, and the electric transfer operation is convenient, but it is necessary to specifically instrument, which is also relatively large for cell damage; viruses
infection overcomes the disadvantages of the top two, infection High efficiency, small toxicity, but the pre-virus pre-packaging needs to be more time and effort.
Experimental operation process:
1. Preparation of transfection reagents
a. 400U to the nucleic acid enzyme water is added to the tube, and 10 seconds, dissolve the lipid.
b. Put the reagent after oscillating - 20 degrees Celsius, it needs to be needed before use.
2. Choose a suitable mixing ratio (1: 1-1: 2 liposome [1 volume: DNA mass to transfection). Add a suitable body
accumulated in a transfection tube. Base. Add appropriate mass of Myod or EGFP ONA, and then adding a suitable volume of transfection reagent, and then shock
.
3, the mixture was placed at room temperature for 10-15 minutes.
4, insert the medium in the culture plate, wash - u0026 nbsp using PBS or serum-free medium; - times.
5, the mixture was added, and the cells were placed back into the incubator - hour.
6 After that, after the cell species decided whether the mixture was removed, it was added to the complete medium to continue to cultivate 24- 48 hours.