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Method for extracting bacillus subtilis genetically engineered bacterium plasmid

A technology of Bacillus subtilis and genetically engineered bacteria, which is applied in the field of extracting plasmids of Bacillus subtilis genetically engineered bacteria, can solve the problems of low quality of plasmid DNA, incomplete wall breaking, and poor repeatability, and achieve good repeatability, complete structure, and easy operation. simple effect

Inactive Publication Date: 2013-04-03
SHANGHAI ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a method for extracting the plasmid of Bacillus subtilis genetically engineered bacteria, so as to solve the defects of incomplete wall breaking, low quality of plasmid DNA, low yield and poor repeatability in the existing extraction methods.

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  • Method for extracting bacillus subtilis genetically engineered bacterium plasmid
  • Method for extracting bacillus subtilis genetically engineered bacterium plasmid
  • Method for extracting bacillus subtilis genetically engineered bacterium plasmid

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Embodiment 1

[0031] 1. Isolation and extraction of Bacillus subtilis plasmid DNA

[0032] 1) Cultivation and collection of Bacillus subtilis: Pick a single colony of Bacillus subtilis strains that have been transformed with different plasmids, culture them in 5ml LB liquid medium with shaking at 30°C for 15 hours, transfer 1 wt% to 50ml fresh LB liquid Continue shaking culture in the medium until the bacterial growth density is OD 600 =0.9-1.1, collect 5ml of bacteria in a 1.5ml centrifuge tube.

[0033] 2) Preparation of protozoa: Add 1.2ml of pre-cooled washing solution A to the collected cells to suspend the cells, centrifuge at 12000g at 4°C for 5min, discard the supernatant, resuspend the cells in 200μl of cell lysate B, and incubate at 37°C 1.5-2h.

[0034] 3) Lysis of cells and extraction of plasmids: Add 300 μl of solution C to the prepared above solution, mix slowly, treat at 68° C. for 10 minutes, and lyse the cells. Add 150μl solution D to the lysate, mix gently, put at -20°C...

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Abstract

The invention discloses a method for extracting bacillus subtilis genetically engineered bacterium plasmid. The method comprises the following steps: step 1) culturing and collecting bacillus subtilis thalli; step 2) preparing a protobiont; step 3) splitting and extracting cells; and step 4) purifying the plasmid. The plasmid DNA (deoxyribose nucleic acid) which is obtained through the method can be directly used for various molecular biologic experiments such as restriction enzyme digestion and PCR (polymerase chain reaction) amplification. The method is simple and safe to operate and high in quality, and can be suitable for extracting plasmid DNA of various gram positive bacteria, so that the method has broad applicability.

Description

technical field [0001] The invention relates to a method for extracting the plasmid of the Bacillus subtilis genetically engineered bacteria. Background technique [0002] Bacillus subtilis (B.subtilis for short) is a species of Bacillus, Bacillus subtilis is a Gram-positive bacterium, which is widely distributed in nature and is an important industrial microorganism. The record of the application of .subtilis is the use of a variant of this species, B.subtilis var.natto (formerly known as Bacillus natto) to ferment soybeans in order to produce bacterial fermented food "Natto (Natto)". This food is a traditional fermented food in Asian countries, and in China, this fermented food is called "douchi". One of the main application areas of Bacillus subtilis enzymes is the food industry. The enzymes used in the food industry in the world account for about 60% of the total, and in my country it is as high as more than 85%. Because its cell wall does not contain endotoxin, it is ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12R1/125
Inventor 王金斌唐雪明李文陈大超祝子坪何建华蒋玮白蓝刘华李鹏吴潇
Owner SHANGHAI ACAD OF AGRI SCI
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