Instruction manual for eukaryotic cell transfection reagents, instructions for using high-efficiency transfection reagents
The EZ Trans cell transfection reagent, as a new generation transfection reagent, is based on the transfection principle of positively charged cationic polymers and negatively charged phosphate groups in nucleic acids forming a positively charged complex, which then interacts with negatively charged proteoglycans on the cell surface and enters the cell through endocytosis. This product has been optimized by Li Ji Biotechnology and has the characteristics of high transfection efficiency, low cytotoxicity, simple operation, good repeatability, and wide applicability.
The following is the instruction manual for eukaryotic cell transfection reagents:
Transportation and storage of transfection reagents
Blue ice transportation. Stored at 4 ℃, with a shelf life of 12 months. [Note]: Cannot be frozen!
Efficient transfection reagent usage method (taking 24 well plate as an example, please refer to Table 1 for transfection in other culture dishes)
1. Inoculate cells
For adherent cells, plate them 18-24 hours before transfection (without antibiotics) to achieve a density of approximately 80% during transfection. For suspended cells, on the day of transfection, plate them before preparing the EZ Trans DNA complex, and add 4-8 × 105 cells to every 500 µ L of growth medium.
[Note]: Serum in the culture medium does not affect transfection efficiency. The amount of transfection reagent used is affected by cell type and other experimental conditions. It is recommended to set a gradient during initial use to optimize the optimal amount used.
2. Prepare EZ Trans DNA complex
(1) For each well of cells, dilute 1 μ g of plasmid DNA to 40 μ L of serum-free high glucose DMEM medium (or OPTI-MEM Ⅰ medium) and mix well.
(2) For each well of cells, dilute 3 μ L of EZ Trans transfection reagent to 40 μ L of serum-free high glucose DMEM medium (or OPTI-MEM Ⅰ medium) and mix gently.
[Note]: Serum free high glucose DMEM medium is a diluent and cannot be used to dilute DNA and EZ Trans transfection reagents with serum containing medium. Because the formation process of EZ Trans DNA transfection complexes cannot contain serum.
(3) Add the diluted EZ Trans transfection reagent to the diluted plasmid DNA as soon as possible and mix gently.
[Note]: The order of this mixing cannot be reversed.
(4) Leave at room temperature for 10-15 minutes to form an EZ Trans DNA complex.
3. Transfect cells
(1) Evenly drop the above 80 μ L EZ Trans DNA transfection complex into a culture dish containing cells. Gently shake the culture dish or shake gently to disperse the EZ Trans DNA complex evenly.
(2) Cultivate in a 5% CO2 incubator at 37 ℃ for 6-18 hours, remove the culture medium containing EZ Trans DNA complex, replace with a new culture medium, and continue to culture until the transferred gene expression analysis is achieved.
[Note]: Select stable transfected cell lines, and after 24 hours of transfection, passage the cells to fresh growth medium (dilute the cells more than 10 times), incubate in a 37 ℃, 5% CO2 incubator for 24 hours, and add the screening medium. Under drug screening conditions matched with transfected resistance genes, resistant clones can be screened in about 1-2 weeks, during which the growth medium containing the screened drug needs to be frequently replaced.
matters needing attention
This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
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