What is cacl2 transfection?
The calcium phosphate transfection method for introducing DNA into mammalian cells is based on forming a calcium phosphate-DNA precipitate. The DNA is mixed directly with a concentrated solution of CaCl2, which is then added dropwise to a phosphate buffer to form a fine precipitate.
How is calcium phosphate used for DNA transfer in animals?
The calcium phosphate transfection is a widely used method for introducing foreign DNA plasmids into cells. Mechanisms underlying this transfection method are not yet defined; however, DNA-calcium phosphate precipitates are internalized by the cells and DNA is efficiently expressed in almost all cell types.
Why do you think the DNA CaCl mix was made separately and then added to phosphate buffer?
Aeration of the phosphate buffer while adding the DNA-calcium chloride solution helps to ensure that the precipitate that forms is as fine as possible, which is important because clumped DNA will not adhere to or enter the cell as efficiently.
Why is transfection important?
Transfection is a powerful analytical tool for study of gene function and regulation and protein function. 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 .
What is the pH of calcium phosphate?
The results show that although calcium phosphate as such can reduce acidity, their buffering range lies at a pH of about 4, i.e. outside the physiological range. This is not related to the rate of dissolution. Carbonated calcium phosphates as well as calcium carbonate (calcite) alone are able to keep the pH around 7.4.
What is transfection in virus?
Virus-mediated transfection, also known as transduction, offers a means to reach hard-to-transfect cell types for protein overexpression or knockdown, and it is the most commonly used method in clinical research (Glover et al., 2005; Pfeifer and Verma, 2001).
How is calcium phosphate used in cell transfection?
Protocol for cell transfection using the calcium phosphate method. Utilize the following mix components for all experiments. This protocol has been optimized for transfection of neonatal rat cardiac myocytes.
How to do calcium phosphate transfection on 24 well plates?
For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid.
How to transfect calcium phosphate with Eppendorf tube?
Combine the above three reagents in a hood using a sterile 1.5 mL Eppendorf tube. Mix gently. Do not vortex. Add 200 μL drop-wise with gentle agitation of the above mixture (setting of 2 on vortex mixer). 300 μl added dropwise with gentle agitation. Then invert several times to mix completely.
How to apply transfection mix to reporter cells?
When ready to apply the mix to cells, combine equal amounts of the reporter plasmid with beta-gal control mix. Mix gently by inverting several times and add 40 μl of the mix to each well of a 24 well plate (this will require optimization depending on the specific experiment) swirling the cells gently to disperse the transfection mix into the media.