Introduction
Calcium phosphate transfection is a generally used methodology for the introduction of DNA into eukaryotic cells. This system has been used to acquire each transient1 and stable2 transfections in all kinds of cell varieties. The process relies on gradual mixing of HEPES-buffered saline containing sodium phosphate with a CaCl2 answer containing the DNA. A DNA−calcium phosphate co-precipitate varieties, which adheres to the cell floor and is taken up by the cell, presumably by endocytosis. Glycerol shock could improve the uptake of DNA in some cell varieties.
Reagents offered
The reagents equipped on this equipment are sterilized by 0.2 µm filter and aseptically crammed. The equipment permits for both:
The Calcium Phosphate Transfection Equipment accommodates the next:
Storage
All elements needs to be saved at –20 °C
Enable all equipment elements to thaw and equilibrate to room temperature earlier than use.
Process
The process acknowledged beneath is designed for the transfection of CHO cells with 1 µg/µl pSV40-CAT plasmid (diluted in sterile molecular biology grade water). Tradition cells in customary serum-containing or serum-free medium acceptable for the cell sort. Antibiotics are usually not really helpful. Use good aseptic approach and use solely sterile supplies.
DNA plasmids needs to be high-quality, ethanol-precipitated, resuspended in molecular biology grade water to a last focus of 1 ug/uL.
This protocol might be optimized to be used with all kinds of cell varieties. Seeding density, quantity of DNA used, incubation time and glycerol shock can simply be diverse to attain larger expression and decrease toxicity when wanted.
Day One: Plate Cells
Plate the cells in line with the next chart:
Day Two: Transfection
Day Three: Optionally available Glycerol Shock
Day Three: Change Medium
Supplies – “calcium phosphate transfection”
References
Associated Content material