This experiment is mainly used to obtain eukaryotic cells containing the target exogenous gene.
Operation method
Stability screening assay for transfected cells
Principle
Exogenous genes are integrated into genomic DNA after transfection of eukaryotic cells and are able to persist in the cell for long periods of time and are passed on to the offspring with chromosome replication. It generally takes several days for an exogenous gene to enter a cultured cell before it can be integrated into genomic DNA.
Materials and Instruments
Transfected Cells Move 1. Determine the optimal concentration of antibiotic action: Different cell lines have different sensitivities to various antibiotics, so it is necessary to do pre-tests before screening to determine the lowest concentration of antibiotics on the selected cells. (1) Inoculate 8 wells of cells in 96-well or 24-well plates 24 hours in advance, with the amount of inoculum appropriate for the next day to grow into a 25% monolayer, and incubate overnight at 37℃ in a CO2 incubator. (2) Replace the culture medium with antibiotic-containing medium, and increase the antibiotic concentration in a gradient (0, 50, 100, 200, 400, 600, 800 and 1000ug/ml). (3) Cultivate for 10-14 days to the concentration of the majority of cell death, generally 400-800ug/ml, when screening stable expression clones can be increased by one level than this concentration to maintain half of the screening concentration. 2. Transfection is carried out according to the previous steps. 3. 72 hours after transfection, the transfected cells are passaged in a 6-well plate at a ratio of 1:10, and replaced by a selection medium containing the antibiotic concentration determined in the pre-test. A single cell can be seen in the 6-well plate, and if the culture is continued, a single cell can divide and multiply to form a single resistant colony, at which point two methods can be used to select the monoclonal. 1) Filter paper method: Use sterilized 5x5mm filter paper soaked in trypsin, stick the filter paper on the single cell colony for 10-15 seconds, take out the filter paper with adhered cells and put it into a 24-well plate to continue to pressurize the culture. After the cells are full grown in the 24-well plate, transfer them to 25cm2 culture flasks, and then transfer them to 75cm2 culture flasks after they are full grown. (2) Limited dilution method: After the cells are digested and serially diluted 10-fold ( 10-2-10-10 ), each dilution of cells is added dropwise to a 96-well plate and cultured. 7-10 days later, the wells in which the single clone grows are selected for cloning. 4) ELISA or Western blotting method: the cells are digested and serially diluted 10-fold (10-2-10-10). 4. ELISA or Western blot to detect the expression of exogenous proteins in monoclonal cells Since the expression level of different clones varies, multiple clones can be selected at the same time to choose the clone with the highest expression to pass on and preserve the seed. Caveat 1. For adherent growth cells, it is generally required that on the day before transfection, trypsin must be applied to treat the cells into single-cell suspensions and re-inoculate them in culture dishes or flasks, and the cell density on the day of transfection should be 70-90% (for adherent cells) or 2 x 106-4×106The cell density on the day of transfection should be 70-90% (adherent cells) or 2×10 6 -4×10 6 cells/ml (suspension cells), and it is best to change the fresh culture medium once 4h before transfection. 2. The plasmid DNA used for transfection must be free of protein, RNA and other chemical contamination, and the OD260/280 ratio should be above 1. 8. For more product details, please visit Aladdin Scientific website.
Antibiotics Culture medium Trypsin
6-well plate 24-well plate 96-well plate CO2 incubator Filter paper sheets Culture flasks