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Method for extracting bacteria plasmid DNA

A bacterial plasmid and bacteria technology, applied in the field of molecular biology, can solve problems such as harming human health, polluting the environment, and taking a long time, and achieve the effects of reducing pollution, reducing harm, and being easy to operate.

Inactive Publication Date: 2008-08-06
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the methods commonly used to extract plasmid DNA from bacteria include boiling method, SDS method, alkali lysis method, etc. These methods are usually extracted from E. The use of toxic substances such as phenol and chloroform not only pollutes the environment but also endangers human health

Method used

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  • Method for extracting bacteria plasmid DNA

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Embodiment 1

[0025] Pick a single colony of Gordonia ZCG2 cultured on a slant and place it in a common bacterial liquid medium for 12 hours with shaking at 25°C, then take 3.0 mL of the bacteria liquid, centrifuge at 9000 rpm for 70 seconds, and wash twice with 2.0 mL of STE solution. Centrifuge at 10,000 rpm for 30 seconds; first add 150 μL solution I and 50 μL lysozyme (purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.), bathe in water at 37°C for 40 minutes, then add 400 μL solution II, and ice-bath for 5 minutes; add 300 μL solution III , ice bath for 20 minutes, 4°C, centrifuge at 12,000rpm for 10 minutes; take the supernatant, add an equal volume of isopropanol, leave it at room temperature for 5 minutes, and centrifuge at 4°C, 12,000rpm for 5 minutes; 7.5mol·L -1 NH 4 Ac and 5mol L -1 LiCl, ice bath for 5 minutes, 4°C, centrifuge at 12000rpm for 5 minutes; take the supernatant, add an equal volume of isopropanol to the supernatant, leave it at room tempera...

Embodiment 2

[0028]Pick a single colony of Pseudomonas B61 cultured on a slant and culture it in a common bacterial liquid medium at 28°C for 18 hours, then take 2.0 mL of the bacterial liquid, centrifuge at 10,000 rpm for 45 seconds, and wash twice with 1.5 mL of STE solution , centrifuge at 10000rpm for 30 seconds; first add 135μL solution I, then add 270μL solution II, ice bath for 4 minutes; add 200μL solution III, ice bath for 20 minutes, 4℃, centrifuge at 12000rpm for 10 minutes; take the supernatant, add an equal volume of iso Propanol, place at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes at 4°C; dissolve the precipitate with 200 μL of sterile distilled water, add 100 μL of 7.5mol L -1 NH 4 Ac and 5mol L 1 LiCl, ice bath for 5 minutes, 4°C, centrifuge at 12000rpm for 5 minutes; take the supernatant, add an equal volume of isopropanol, and let it stand at room temperature for 40 minutes, the precipitate is plasmid DNA; wash the above precipitate twice wit...

Embodiment 3

[0031] Pick a single colony of Pallidinus humanis ZHL4 cultured on a slant and culture it in a common bacterial liquid medium at 30°C for 20 hours, then take 1.5mL of the bacterial liquid, centrifuge at 11000rpm for 35 seconds, wash twice with 1.0mL of STE solution, 10000rpm Centrifuge for 30 seconds; first add 100 μL of solution I, then add 200 μL of solution II, ice-bath for 3 minutes; add 150 μL of solution III, ice-bath for 15 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C; take the supernatant, add an equal volume of isopropanol , placed at room temperature for 5 minutes, centrifuged at 12,000 rpm for 5 minutes at 4°C; dissolved the precipitate in 200 μL of sterile distilled water, and added 100 μL of 7.5 mol L -1 NH 4 Ac and 5mol L -1 LiCl, ice bath for 5 minutes, 4°C, centrifuge at 12000rpm for 5 minutes; take the supernatant, add an equal volume of isopropanol, and let it stand at room temperature for 40 minutes, the precipitate is plasmid DNA; wash the abov...

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Abstract

The invention relates to molecular biology, which particularly relates to an extracting method of bacterial plasmid DNA. The extracting method comprises the following steps: jolting and overnight culturing microbial thallus in fluid medium under the temperature which is 25 DEG C-30 DEG C, adding buffer solution in bacterial liquid which is overnight cultured, centrifuging to gather and wash to get cracking thallus, separating and purifying plasmid DNA, directly using plasmid DNA which is got in various molecular biology tests such as restriction enzyme, PCR and the like. The extracting method has the advantages of simple and convenient operation and high quality, and meanwhile, the invention is suitable for the extraction of Gram-negative bacteria and Gram-negative bacteria plasmid DNA, therefore, the extracting method has wide adaptability.

Description

technical field [0001] The invention relates to molecular biology, in particular to a method for extracting bacterial plasmid DNA. Background technique [0002] Plasmid is a circular double-stranded DNA molecule that exists outside the bacterial chromosome and can be independently replicated and inherited stably. Plasmid DNA is a common carrier for genetic engineering. It can carry foreign genes into bacteria, animal cells or plants for amplification and expression. The efficiency and quality of plasmid DNA extraction are directly related to the success of subsequent experiments (enzyme digestion, PCR amplification, etc.), so it is of great significance to quickly and efficiently extract and purify plasmids from bacterial cells. [0003] At present, the methods commonly used to extract plasmid DNA from bacteria include boiling method, SDS method, alkaline lysis method, etc. These methods are usually extracted from E. The use of toxic substances such as phenol and chloroform...

Claims

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Application Information

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IPC IPC(8): C12N15/31
Inventor 郑乐刘宛李培军张利红
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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