Cell transfection FAQ
First, the transfection efficiency is low
There are many factors affecting transfection efficiency . The main factors are cell culture, serum, vector construction, DNA quality and transfection technology .
1. No optimization conditions were used: optimize the amount of cationic liposome reagent and DNA.
2. The DNA-cationic liposome reagent complex is formed in the presence of serum.
3. Presence of inhibitor: Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate in the medium used to prepare the DNA-cationic liposome complex. Hyaluronic acid, dextran sulfate or other proteoglycans.
4. Inappropriate cell density: The degree of fusion should be 70%-90% when transfected.
5. Cationic liposome reagent freezing: Do not use cationic liposome reagents that are frozen or stored at temperatures below 4 °C.
6. Problems with plasmid purification.
Second, high cell death rate
1. The amount of DNA is too high
2. The amount of cationic liposome reagent is too high
3. Use antibiotics during transfection: Do not use chloramphenicol, penicillin or streptomycin during transfection because cationic liposome reagents make cells more sensitive.
4. Too few cells
5. Reduced cell viability in serum-free conditions: using OPTI-MEM medium. Ensure that the complex is formed in the absence of serum.
6. The cationic liposome reagent is oxidized: do not excessively agitate or oscillate the cationic liposome reagent, which may form a peroxide of the cationic liposome reagent.
7. For stable transfection, screening for antibiotics is too fast: at least 24-48h prior to the addition of screening antibiotics to allow cells to express resistance genes.
Cell transfection considerations
1. If adherent cells are grown, it is generally required to apply pancreatin to a single cell suspension on the day before transfection, and re-inoculate the culture dish or bottle. The cell density on the day of transfection is 70-90%. Preferably, the parietal cells are 2 x 106 - 4 x 106 cells/ml (suspended cells), and it is preferred to change the fresh medium by 4 h before transfection.
2. The plasmid DNA used for transfection must be free of protein, free of RNA and other chemicals, and the OD260/280 ratio should be above 1.8.
3. Transfection with serum:
1) Serum-free media should be used at the beginning of preparation of DNA and cationic liposome reagent dilutions, as serum can affect the formation of complexes. In fact, as long as the DNA-cationic liposome complex is free of serum, serum can be used during transfection.
2) The optimal amount of cationic liposome and DNA will vary when using serum, so the conditions need to be optimized when serum is added to the transfection medium.
3) Most cells can remain healthy for several hours in serum-free medium. For cells that are sensitive to serum deficiency, OPTI-MEM medium, a nutrient-rich serum-free medium, can be used.
4. Antibiotics in the medium
Antibiotics are media supplements that affect transfection. These antibiotics are generally non-toxic to eukaryotic cells, but cationic liposome reagents increase the permeability of the cells, allowing antibiotics to enter the cells. This reduces the activity of the cells, resulting in reduced transfection efficiency. Therefore, antibiotics cannot be used in the transfection medium.
For stable transfection, do not use penicillin and streptomycin in selective media because these antibiotics are competitive inhibitors of GENETICIN selective antibiotics, the most commonly used resistance screening agents for stable transfection. In addition, in order to ensure healthy growth of cells in serum-free medium, a smaller amount of antibiotics than serum-containing medium is used.
5. Set up positive and negative controls.
6. Generally, after transfection for 24-48 hours, the target gene is expressed in the cells. According to different experimental purposes, the detection of target gene expression can be performed after 24-48h.
7. If a stable cell line is established, the target cells can be screened, and the corresponding drugs are selected according to the resistance markers contained in the different gene vectors. The commonly used eukaryotic expression gene vectors are labeled with hygromycin and new mold. Prime.
Transfection reagent:
Lipofectamine® 2000 Transfection Reagent (Invitrogen)
DNA Transfection Reagent EntransterTM-H-4000 (Engreen)
RNA Transfection Reagent EntransterTM-R-4000 (Engreen)
ViaFectTM Transfection Reagent (Promega)
FuGENE® HD Transfection Reagent (Promega)
FuGENE® 6 Transfection Reagent (Promega)
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