Method for extraction and purification of plasmid DNA and its preparation process

A plasmid and production method technology, applied in chemical instruments and methods, sugar derivatives, sugar derivatives, etc., can solve the problems of difficult to master the cracking process, difficult to remove pollution, affect efficiency, etc., to achieve easy implementation and production scale-up, overcome Insufficient cracking, the effect of improving yield and quality

Inactive Publication Date: 2004-11-17
CHINA AGRI UNIV
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Problems solved by technology

However, with the rapid development of the research and application of DNA vaccines and gene recombinant drugs, the demand for recombinant plasmids is increasing, and conventional laboratory preparations and methods are difficult to meet; another deficiency is that the use of these two methods not only High cost, low output, poor quality, and difficult to scale up
As a result, the efficiency of later work is affected, and it is currently called a bottleneck in the research and application of DNA vaccines and gene recombinant drugs
At present, there are still some large-scale extraction methods that use various types of affinity chromatography columns. The extraction effect is better but the cost is high, and it is not easy to expand the scale.
Alkaline cracking method needs to add a variety of reagents in the extraction process, and there is reagent pollution in the final product, and the yield and quality are not high; while the boiling cracking method has high yield, but the operation is complicated, the cracking process is difficult to master, the result is unstable, and the pollution is not easy remove
Therefore, at present, the development of many nucleic acid technologies is constrained by the insufficient technology of preparing plasmid DNA in large quantities.

Method used

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  • Method for extraction and purification of plasmid DNA and its preparation process
  • Method for extraction and purification of plasmid DNA and its preparation process
  • Method for extraction and purification of plasmid DNA and its preparation process

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: the fermentation of engineering bacterium

[0044] The plasmid pc3d-N was transformed into XL1-blue competent cells, and a single colony was picked from the AMP agar plate and cultured in 5 ml LB (with appropriate amount of antibiotics) at 37°C until the logarithmic growth phase. Take 1.5 ml of the culture and culture it overnight at 37° C. in 150 ml of LB (plus appropriate amount of antibiotics). Add 150 milliliters of the overnight culture to a fermenter filled with 3 liters of LB medium (plus appropriate amount of antibiotics) for fermentation at 37° C. After fermenting for 5 to 6 hours, feed 400 milliliters. OD after 15h of fermentation 600 When there is no longer a significant increase, stop the fermentation and measure the OD 600 for 45.

Embodiment 2

[0045] Embodiment 2: Thalline collection

[0046] 3.5 L of the fermentation broth was pumped into a hollow fiber membrane column with a peristaltic pump for concentration, and after concentration, the cells were washed with 200 ml of TE (10 mM Tris-HCL, 50 mM EDTA, pH=8.0). After concentration, an equal volume of TE was added to the concentrate to continue concentration, and 3.5 L of fermentation broth was finally concentrated to obtain 350 ml of concentrate.

Embodiment 3

[0047] Embodiment 3: Thalline collection

[0048] 15 L of the fermentation broth was pumped into a hollow fiber membrane column with a peristaltic pump for concentration, and after concentration, the cells were washed with 500 ml of TE (10 mM Tris-HCL, 50 mM EDTA, pH=8.0). After concentration, an equal volume of TE was added to the concentrated solution to continue concentration, and 15 L of fermentation broth was finally concentrated to obtain 750 ml of concentrated solution.

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Abstract

Method for extraction and purification of plasmid DNA from prokaryotes and its preparation process, wherein the process comprises the steps of prokaryote bacterium liquid concentration, splitting thalline, and plasmid DNA purification.

Description

technical field [0001] The present invention relates to a method for extracting and purifying plasmid DNA from prokaryotes and its production process, which is one of the most basic steps in molecular biology and involves gene cloning, gene sequence analysis, nucleic acid vaccines, gene therapy and various genetic recombination technology and other fields. Background technique [0002] The extraction and purification of plasmid DNA is one of the most basic steps in molecular biology, involving the fields of gene cloning, gene sequence analysis, nucleic acid vaccine, gene therapy and various gene recombination. The classic extraction and purification of plasmid DNA from prokaryotes is divided into three steps: cell lysis, plasmid isolation and purification. Currently, there are two most commonly used methods for cell lysis: alkaline lysis (Bimboim and Doly, Nucleic Acids Res., 7: 1513-1523, 1979) and boiling lysis. (Holmes and Quigley, Anal. Bioche...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H1/06C07H21/04C12P19/34
Inventor 王宾俞庆龄
Owner CHINA AGRI UNIV
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