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Kit and method for extracting plasmid DNAs by one step

A technology for extracting plasmids and kits, which is applied in the field of kits for one-step extraction of plasmid DNA, which can solve the problems of low supercoiled plasmid content, long extraction experiment cycle, and large limitations, and achieve rapid and efficient separation and efficient and rapid removal of impurities , time-saving effect

Inactive Publication Date: 2017-10-17
GENFINE BIOTECH BEIJING CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] The purpose of the present invention is to provide a kit and method for one-step extraction of plasmid DNA, which are used to solve the technical problems of long experimental period for extraction of plasmid DNA, low content of supercoiled plasmid in the extract, high cost and large limitations

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  • Kit and method for extracting plasmid DNAs by one step
  • Kit and method for extracting plasmid DNAs by one step

Examples

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Embodiment 1

[0032] Example 1 Extraction of high-copy plasmid pBS from E. coli culture

[0033] ① Amplification of plasmid: take 1.5 mL of E. coli broth cultured in LB medium overnight, centrifuge at 12000r / min for 1 min, discard the supernatant, and obtain the bacterial pellet;

[0034] ②Bacterial cell lysis: add 500μL of pre-cooled lysis reagent to the bacterial cell sediment, vortex for 30s until the bacterial cell sediment is completely dissolved, and obtain the bacterial cell lysate;

[0035] ③Purification of plasmid DNA: Place the cell lysate in step ② at room temperature for 2 minutes, then transfer it to a silicon matrix membrane adsorption column, centrifuge at 12000r / min for 30 seconds, discard the waste liquid; then add it to the silicon matrix membrane adsorption column 300μL rinsing solution, desalted and centrifuged at 12000r / min for 30s, discarded the waste solution; finally, 80μL of elution buffer was added to the silicon matrix membrane adsorption column, and the elution was cent...

Embodiment 2

[0037] Example 2 Extraction of high-copy plasmid pBS from E. coli culture

[0038] ① Amplification of plasmid: Take 1.5 mL of Escherichia coli broth cultured overnight with MUG medium, centrifuge at 12000r / min for 1 min, discard the supernatant, and obtain bacterial pellet;

[0039] ②Bacterial cell lysis: add 500μL of pre-cooled lysis reagent to the bacterial cell sediment, vortex for 30s until the bacterial cell sediment is completely dissolved, and obtain the bacterial cell lysate;

[0040] ③Purification of plasmid DNA: Place the cell lysate in step ② at room temperature for 2 minutes, then transfer it to a silicon matrix membrane adsorption column, centrifuge at 12000r / min for 30 seconds, discard the waste liquid; then add it to the silicon matrix membrane adsorption column 300μL rinsing solution, desalted and centrifuged at 12000r / min for 30s, discarded the waste solution; finally, 80μL of elution buffer was added to the silicon matrix membrane adsorption column, and the elution ...

Embodiment 3

[0042] Example 3 Extraction of low-copy plasmid pBR322 from E. coli culture

[0043] ① Amplification of plasmid: Take 1 mL of Escherichia coli bacteria cultured overnight in LB medium, centrifuge at 12000r / min for 1 min, discard the supernatant, and obtain the bacterial pellet;

[0044] ②Bacterial cell lysis: add 500μL of pre-cooled lysis reagent to the bacterial cell sediment, vortex for 30s until the bacterial cell sediment is completely dissolved, and obtain the bacterial cell lysate;

[0045] ③Purification of plasmid DNA: Place the cell lysate in step ② at room temperature for 2 minutes, then transfer it to a silicon matrix membrane adsorption column, centrifuge at 12000r / min for 30 seconds, discard the waste liquid; then add it to the silicon matrix membrane adsorption column 300μL rinsing solution, desalted and centrifuged at 12000r / min for 30s, discarded the waste solution; finally, 80μL of elution buffer was added to the silicon matrix membrane adsorption column, and the elut...

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Abstract

The invention discloses a kit and a method for extracting plasmid DNAs by one step, and belongs to the field of biotechnology. The invention adopts the technical scheme as follows: the kit for extracting the plasmid DNAs by one step comprises a lysing reagent, a rinsing solution and an eluting buffer solution, wherein the lysing reagent comprises lysozyme, RNase A and a lysing buffer solution. On the basis of the optimized lysing buffer solution and a synergistic lysozyme and RNase A rapid lysing method, one-step bacterial lysis, namely application to a column, is achieved, the plasmid extracting cycle is shortened to 5min, the supercoiled plasmid content is 90% or above, the experimental efficiency is improved, and high-quality plasmids are provided for a subsequent experiment; the kit and the method have the characteristics of high efficiency, rapidness and simplicity.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a kit and method for extracting plasmid DNA in one step. Background technique [0002] Plasmid DNA is the most common carrier of genetic engineering, and it is gradually becoming the most important gene delivery system in gene therapy research. It can send target DNA fragments into recipient cells through recombinant DNA technology for reproduction and expression. The extraction and purification of plasmid DNA is one of the basic steps of molecular biology, involving gene cloning, gene sequence analysis, nucleic acid vaccines, gene therapy, and various gene recombination fields. The extraction efficiency and quality are important for subsequent molecular biology experiments ( Enzyme digestion, PCR amplification and sequencing, etc.) have a close and direct relationship. [0003] Studies have found that the extracted plasmids generally have three conformations: supercoiled, linear and open ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 韩典霖杨亮龚小鹏
Owner GENFINE BIOTECH BEIJING CO LTD
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