How does calcium phosphate transfection work?
Table of Contents
How does calcium phosphate transfection work?
The calcium phosphate transfection method for introducing DNA into mammalian cells is based on forming a calcium phosphate-DNA precipitate. Calcium phosphate facilitates the binding of the DNA to the cell surface. DNA then enters the cell by endocytosis.
What is the purpose of transfection?
The main purpose of transfection is to study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells, and to produce recombinant proteins in mammalian cells [3].
How do you prevent calcium phosphate precipitation in TPN?
The risk of calcium phosphate precipitation may be reduced by:
- Keeping the final amino acid concentration at 2.5% or greater.
- Maintaining a final pH of 6.0 or lower.
- Infusing the solution within 24 hours of preparation.
- Using calcium gluconate instead of calcium chloride.
Should I change media before transfection?
It depends when you plate the cells for the transfection. If you plate the cells one day before transfection, then it may not be necessary to replace the medium. Off course, if you plate cells several days before the transfection, then it may be a good idea to change the medium before transfection.
How do you know if a transfection was successful?
Determining the number of positive cells within a transfected cell population can be done through microscopy and flow cytometry. Finally, confirming localization of your protein of interest can be done by microscopy.
When can I harvest after transfection?
24–72 hours
Cells are typically harvested 24–72 hours after transfection for studies designed to analyze transient expression of transfected genes. The optimal time interval depends on the cell type, research goals and specific expression characteristics of the transferred gene.
How much DNA do you add to transfection?
Optimal amount of Universal Transfection Reagent used depends on cell type and is generally 1 – 3 µL per ug of plasmid DNA.
