There are many factors affecting transfection, and the main factors are as follows.
First, the state of the cell
Before starting to transfect cells, develop an appropriate seeding protocol to maintain cell density from the beginning to the end of transfection. Generally low cell algebra (<50) ensures that the genotype remains unchanged. The most suitable cells for transfection are cells that reach the exponential growth phase after several passages. The cells grow vigorously and are most easily transfected.
Second, DNA quality
DNA quality has a significant impact on transfection efficiency. General transfection techniques, such as liposomes, based on the principle of charge attraction, if the DNA is impure, a small amount of salt ions, protein, metabolite contamination can significantly affect the effective formation and transfection of the transfection complex. In addition, some endotoxin-sensitive cells (such as primary cells, suspension cells and hematopoietic cells) need to effectively remove lipopolysaccharide molecules during plasmid extraction to ensure the desired transfection effect.
Third, the transfection method
Different cells are suitable for different transfection methods. If the laboratory has established a complete set of methods and found the best transfection conditions, it will be done. If it is a new cell line, it is best to look at its successful transfection case. Refer to the list of cell lines that have been successfully transfected by the transfection reagents and literature. If your cells are suitable, do so according to the recommended method of the selected transfection reagent. Many transfection methods require optimization of DNA to transfection reagent ratio, cell number, culture and detection time. ViaFect Transfection Reagent is a new generation transfection reagent with an innovative mixed lipid formulation for DNA transfection, high efficiency and low toxicity. There is no need to remove serum, and after introduction of the transfection reagent: DNA complex, there is no need to wash or change the medium, which is the recommended choice for expression experiments of the target gene in cells.
Examples of ViaFect transfection reagents for adherent cell transfection procedures:
1. Spread the cells one day before transfection, and the cell confluence is about 75% on the day of transfection.
2. On the day of transfection, the DNA will be diluted in serum-free medium. Add appropriate amount of ViaFect transfection reagent to the appropriate ratio of DNA and incubate for 5-20 min.
3. Add appropriate amount of transfection reagent: DNA mixture to the cell culture well and mix gently.
4. There is no need to remove serum or change culture conditions. Incubate for 24-48 hours or appropriate time to test the results. There is no need to remove the transfection mixture.
5. Test the experimental results.
Fourth, transfection reagent
The transfection efficiency of different cell lines is usually different, but the selection of cell lines is usually based on the needs of the experiment. Therefore, suitable transfection reagents should be selected according to experimental requirements and cell characteristics before transfection experiments. Each transfection reagent provides a list of cells that have been successfully transfected and literature, and the selection of transfection reagents that are best suited for the experimental design. Of course, the most suitable is a highly efficient, low toxicity, convenient and inexpensive transfection reagent. For example, ViaFect transfection reagents are suitable for many types of cell lines and successfully transfected into a variety of types, including adherent cells, suspension cells and stem cell-derived cells, which are more efficiently transfected in a variety of difficult-to-transfect cells. In addition, ViaFect transfection reagent has low toxicity and can maintain the biological and cellular metabolism of cells during transfection, thereby more accurately presenting the biological state of the cell model. Moreover, ViaFect transfection reagents are easy to use and can be reverse transfected without complex optimization. It can be said that among similar transfection reagents, ViaFect transfection reagent is more cost-effective. (Note: Reverse transfection refers to the method of first adding DNA to the wells of the culture plate: transfection reagent mixture, and then adding cells for transfection)
name | Catalog number (click to view) | specification |
ViaFect Transfection Reagent | E4981 | 0.75ml |
E4982 | 2×0.75ml | |
E4983 | 0.2ml |
Fifth, the carrier construction
Construction of the transfected vector (viral vector, plasmid DNA, RNA, PCR products, oligonucleotides, etc.) also affects the transfection results. Viral vectors are more efficient at infecting specific host cells, but different viral vectors have their own specific hosts, and some require specific cell cycles, such as retroviruses that need to infect mitotic host cells, and some safety issues need to be considered. (such as genetic contamination). In addition to vector construction, the morphology and size of the vector also have different effects on transfection efficiency. If the gene product is toxic to the cell, transfection is also difficult to perform, so it is important to select a promoter that is constitutive or regulatable and of suitable strength. At the same time, the transfection of the same vector constructed with the empty vector and other genes can eliminate the toxicity. Impact of interference.
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