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Method for transfecting cells with calcium phosphate

A technology for transfecting cells and calcium phosphate, applied in the field of cell culture, can solve the problems of low transfection efficiency, many operation steps, low transfection efficiency, etc., and achieve the effect of improving transfection efficiency, simple operation steps and good repeatability.

Inactive Publication Date: 2019-09-13
THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
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  • Summary
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  • Application Information

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Problems solved by technology

However, with the wide application of this technology, it has been found that in the calcium phosphate transfection method, there are many factors that affect the success of transfection and the level of transfection efficiency, often resulting in low efficiency and instability of transfection.
There are many operating steps and long time-consuming, often resulting in low transfection efficiency and instability

Method used

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  • Method for transfecting cells with calcium phosphate

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Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1, the method for calcium phosphate transfection 293T cell

[0030] 1. Experimental method

[0031] (1) 293T cells were collected by trypsinization before transfection, and the transfection could begin when the area occupied by the cells reached 60%-70% of the total area of ​​the culture dish after culturing;

[0032] (2) Preparation of calcium phosphate-plasmid DNA precipitation: Take an EP tube, add 10 μg each of recombinant shuttle and backbone plasmid, and then add Buffer B (Cacl 2 ) 50 μl (10×), and finally make up the system to 500 μl with sterilized water, and mix gently. The use concentration of the BufferB is 2.5mol / L.

[0033] (3) Take another 50ml round-bottomed test tube, add Buffer B (500μl) of the same volume as above, blow and bubble Buffer B from the bottom with a straw (bubble as large as possible), gradually and slowly mix the ingredients in step 2 Add the good mixture dropwise to Buffer B, this step should be completed within 2 minutes a...

Embodiment 2

[0041] The present invention also carried out the method of transfecting 293T cells with calcium phosphate under other conditions with reference to Example 1, and carried out experimental observations.

[0042] Condition 1: The difference from Example 1 is that the concentration of Buffer B used is 3 mol / L, and in step (6), the medium is replaced 18 hours after transfection. Everything else is the same.

[0043] Condition 2: The difference from Example 1 is that the concentration of Buffer B used is 2.7mol / L. In step (6), the medium was changed 24 hours after transfection. Everything else is the same.

[0044] Microscopic observation results showed that the method of transfecting 293T cells with calcium phosphate in the above two conditions had a good transfection effect, and the transfection efficiency was between 80% and 85%.

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Abstract

The invention relates to a method for transfecting cells with calcium phosphate. The method at least comprises the following steps of: 1) inoculating cells into a culture container containing a culture medium for adherent culture, and starting transfection when the cells account for 60-70% of the total area of the wall of the adherent culture container so as to obtain cells to be transfected; 2) preparing calcium phosphate-plasmid DNA precipitation: adding CaCl2 aqueous solution into the plasmid, adding sterile water, and performing uniform mixing to obtain a mixed solution I; 3) blowing and foaming the CaCl2 aqueous solution with the same volume as the mixed solution I to obtain a foam mixed solution II; 4) adding the mixed solution I into the foam mixed solution II, and performing incubating at room temperature to obtain a mixed solution III; 5) replacing a fresh culture medium for the cells to be transfected, and performing standing; and 6) adding the mixed liquid III into the culture medium in which the cells to be transfected are located, performing uniform mixing, and performing incubating. The method improves the transfection efficiency, and has the characteristics of simpleoperation steps, reliable results, good repeatability and the like.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for transfecting cells with calcium phosphate. Background technique [0002] There are many ways to introduce DNA into animal cells. The calcium phosphate transfection method is economical, simple and non-toxic to cells. It is currently one of the most commonly used methods for eukaryotic cell transfection. This method is used as a routine method for gene transfection of various cells, and can obtain transient or long-term expression. However, with the wide application of this technology, it has been found that in the calcium phosphate transfection method, there are many factors that affect the success of transfection and the level of transfection efficiency, often resulting in low efficiency and instability of transfection. There are many operating steps and long time-consuming, often resulting in low and unstable transfection efficiency. [0003] How...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
CPCC12N15/63
Inventor 王小文胡嘉彪黄春蒋迎九吴庆琛
Owner THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
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