Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for separating and purifying large plasmids from bacteria

A technology for separation and purification of bacteria, applied in the fields of biochemistry and molecular biology, to achieve effective extraction

Inactive Publication Date: 2012-07-04
JIANGSU UNIV
View PDF0 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to overcome the deficiency that the traditional alkaline lysis method cannot be used to extract large bacterial plasmids, the present invention provides a simple and practical method for isolating and purifying large plasmids from bacteria

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for separating and purifying large plasmids from bacteria
  • Method for separating and purifying large plasmids from bacteria
  • Method for separating and purifying large plasmids from bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0020] Example: Take the isolation and purification of Salmonella typhi first-line plasmid pBSSB1 (27kb) as an example:

[0021] The specific process is:

[0022] (1) Cultivate bacteria: pick Salmonella typhi GIFU10007 ( S . Typhi) was inoculated in 4ml LB liquid medium, cultured overnight at 37°C with shaking (250rpm) and then centrifuged (4000rpm, 10min, 4°C) to collect the bacteria.

[0023] (2) Make gel blocks: After washing with PIV buffer (10mM Tris, 1M NaCl, PH7.6), take 1ml of PIV buffer to resuspend the bacteria, and mix with 1ml of 2% low-melting point agarose gel (keep the temperature At 50°C), pour it into the glue block pouring tank to make Salmonella typhi glue blocks.

[0024] (3) Digest protein: put Salmonella typhi gel into fresh Lysis buffer (6mM Tris, 0.1M EDTA, 1M NaCl, 0.5% Brij-58, 0.2% sodium deoxycholate, 0.5% SDS, RNaseA 20μg / ml, lysozyme 1mg / ml) at 37°C for overnight shaking, ES buffer (0.5M EDTA, 1% SDS) to wash the module and then digest with ES...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for separating and purifying large plasmids from bacteria, and belongs to the technical fields of biochemistry and molecular biology. The method comprises the following steps of: embedding the bacteria in a low smelting point agarose gel; breaking the bacteria in the gel by using lysozyme, and digesting the protein of the bacterial in the gel by using proteinase; separating the DNA of the bacteria by using pulsed field gel electrophoresis; and recovering the gel to obtain the large plasmid DNA in the bacteria. The large plasmid DNA obtained through separation and purification has the characteristics of high purity, complete structure and the like. The method has important significance for researching biological characteristics and functions of the large plasmids and transforming and developing and utilizing the large plasmids in the bacteria.

Description

technical field [0001] A method for separating and purifying a large plasmid from bacteria belongs to the technical field of biochemistry and molecular biology. It can be used for the extraction and identification of large bacterial plasmids. Background technique [0002] Plasmids are extrachromosomal self-replicating genetic elements ranging in size from 1 kb to over 200 kb, widely present in cells of various organisms, especially in bacteria, encoding certain organisms that are not necessary for the survival of bacteria Chemical traits, such as sexual pili, bacteriocins, toxins, and drug resistance, are generally not integrated into bacterial chromosomes. Most of the commonly used plasmids are modified or artificially constructed, often containing antibiotic resistance genes, and are important tools in recombinant DNA technology in molecular biology. Isolation and extraction of plasmids in bacteria is the first step in the research and transformation of plasmids. For sma...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 张海方黄新祥倪斌杜鸿许化溪徐顺高生秀梅
Owner JIANGSU UNIV