Cell transient transfection
Transient transfection of cells can be used to (1) observe the function of a target gene (2) express the function of a protein in a certain cell (observations can be made in a short period of time, but the effect is quickly lost).
Operation method
Cell transient transfection
Principle
Through liposome-mediated transfection and gene transfection techniques such as electroporation, target genes are introduced into the cells, and generally the target genes can be expressed in the cells about 48 hours after transfection. Depending on the purpose of the experiment, the target gene expression can be detected after 48 hours.
Materials and Instruments
Cell Samples Move General process: 1) Cell inoculation: cells are inoculated the day before the transfection experiment, and the plate density of various cells is based on the growth rate and cell shape of various cells. Cell density should be 60%~80% covered on the day of conducting transfection. 2) Cell transfection (liposome, electroporation, FuGENE6, etc.) 3) Identification of target gene expression after 48 hours. Pre-preparation: l Constructed exogenous gene vector, or basic information of the target gene. l The cell line to be transfected (1×106 cells, can be passed on, multiplication time less than 48 hours) and the description of cell culture conditions. l If you need to detect the expression of the target gene, please provide the corresponding antibody. Caveat 1. Gene expression can be detected 48--72h after transfection. It can be used for recombinant protein, antibody production, etc. 2. Because the details of each experiment may be different, it is recommended to do optimization experiments to determine the optimal ratio of sinofection transfection reagents to DNA before transfection. 3. If transfection is performed under serum-free conditions, use normal serum-containing growth medium for cell spreading. Remove the growth medium and replace it with 0.5 ml of serum-free medium before adding the complex. Common Problems 24-72 hours after addition of the complex to the cells, cell extracts were analyzed or in situ cell staining was performed to detect reporter gene activity. This is dependent on cell type and promoter activity. For stable expression, cells are passaged into fresh medium one day after starting transfection and screening antibiotics are added two days later. Several days or weeks are required to perform stable expression. For more product details, please visit Aladdin Scientific website.
FuGENE 6 Liposomes
Electroporation