Calcium phosphate transfection (CAT#: STEM-GT-0004-WXH)

Introduction

The calcium phosphate transfection is an inexpensive and simple method for transient or stable nucleotide transfer into most cell lines. The transfection efficiency, however, strongly depends on the cell constitution, the pH, and the quality and the amount of the used nucleotides. Therefore, the optimal experimental conditions have to be tediously established in advance. Further, the calcium phosphate transfection is toxic and therefore not suitable for most sensitive primary cell lines.

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Principle Applications Procedure Materials Notes Related Products

Principle

A mixture of the nucleotides, calcium, and phosphate buffer forms a precipitate that is taken up by the cells via endocytosis. Then, the nucleotides either escape the endosome or undergo lysosomal degradation, of which the latter might lead to reduced transfection efficiency. Successfully escaped nucleotides can then be expressed in the target cells.

Applications

Transient and stable transfections

Procedure

1. Mix DNA with calcium chloride and add in a controlled manner to a buffered saline/phosphate solution.
2. Incubate at room temperature to generate a precipitate of extremely small, insoluble particles containing condensed DNA.
3. Add the DNA-calcium phosphate co-precipitate to cells, which adhere to the cell membrane.
4. Assay cells for transient gene expression or select for stable transfection.

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