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Extraction method for DNA of plasmids of escherichia coli

A technology of Escherichia coli and extraction method, which is applied in the field of plasmid extraction, can solve the problems of low supercoiled plasmid content, low effective plasmid content, and low bacterial liquid expression, and achieve short time consumption, increase plasmid content, and scientific and reasonable process Effect

Inactive Publication Date: 2019-02-15
安徽欣伯玉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the cell lysis step mostly adopts the alkali lysis method. The plasmid DNA purification technology of the alkali lysis method was invented by Birnboim&Doly in 1979. Its operation includes three kinds of solution treatment, which requires the first step to suspend the bacteria, and the second step to denature with alkali and SDS Cleavage and the third step of plasmid renaturation, and then to the spin column, the extraction process generally takes about 20 minutes, the experimental cycle is relatively long, and the content of supercoiled plasmid is low, which affects the experimental progress of researchers
Moreover, the extraction efficiency of the existing plasmid DNA is low, the expression level of the bacterial solution is very low, and the content of the extracted effective plasmid is also very low, which seriously limits the application of the plasmid DNA.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A method for extracting Escherichia coli plasmid DNA, comprising the steps of:

[0033] (1) Purification culture:

[0034] Select Escherichia coli DH5α strain, inoculate the selected DH5α strain on the plate of LB solid medium containing ampicillin by streaking on the plate, after standing for 20 minutes, seal the inoculated plate and place it upside down in the incubator for cultivation ;

[0035] (2) Expansion of training:

[0036] When a single colony grows on the plate in step (1), and the size of the colony reaches 0.5mm, take out the plate, pick a single colony and inoculate it in 4L LB liquid medium containing ampicillin, and then inoculate the LB liquid medium Place it in a shaker for cultivation, and carry out alternating light and sound waves while cultivating;

[0037] (3) Preparation of premix:

[0038] Put solution Ⅰ and solution Ⅱ into a sterile centrifuge tube at a volume ratio of 1:1.6, shake well, place on ice for 4 minutes, and then measure solutio...

Embodiment 2

[0054] A method for extracting Escherichia coli plasmid DNA, comprising the steps of:

[0055] (1) Purification culture:

[0056] Select Escherichia coli DH5α strain, and inoculate the selected DH5α strain on the plate of LB solid medium containing ampicillin by streaking on the plate. After standing for 25 minutes, seal the inoculated plate and place it upside down in the incubator for cultivation ;

[0057] (2) Expansion of training:

[0058] When a single colony grows on the plate in step (1), and the size of the colony reaches 0.55mm, take out the plate, pick a single colony and inoculate it in 4.5L LB liquid medium containing ampicillin, and then culture the LB liquid after inoculation The base is cultured in a shaker, and the light wave and sound wave are alternately treated while culturing;

[0059] (3) Preparation of premix:

[0060] Put solution Ⅰ and solution Ⅱ into a sterile centrifuge tube at a volume ratio of 1:1.8, shake well, place on ice for 5 minutes, and ...

Embodiment 3

[0076] A method for extracting Escherichia coli plasmid DNA, comprising the steps of:

[0077] (1) Purification culture:

[0078] Select Escherichia coli DH5α strain, inoculate the selected DH5α strain on the plate of LB solid medium containing ampicillin by streaking on the plate, after standing for 30 minutes, seal the inoculated plate and place it upside down in the incubator for cultivation ;

[0079] (2) Expansion of training:

[0080] When a single colony grows on the plate in step (1), and the size of the colony reaches 0.6mm, take out the plate, pick a single colony and inoculate it in 5L LB liquid medium containing ampicillin, and then inoculate the LB liquid medium Place it in a shaker for cultivation, and carry out alternating light and sound waves while cultivating;

[0081] (3) Preparation of premix:

[0082] Put solution Ⅰ and solution Ⅱ into a sterile centrifuge tube at a volume ratio of 1:2, shake well, place on ice for 6 minutes, and then measure solution ...

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PUM

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Abstract

The invention discloses an extraction method for DNA of plasmids of escherichia coli. The extraction method comprises the following steps: (1) purification and cultivation; (2) amplified cultivation;(3) preparation of a premixed solution; (4) splitting of escherichia coli; (5) separation of DNA of plasmids; and (6) collection of the DNA of plasmids. The invention provides the extraction method for DNA of plasmids of escherichia coli. The whole process is scientific and reasonable. The contents of plasmids in a unit bacterial solution and DNA of superhelix plasmids in unit mass plasmids are improved. The time consumed is short, and the market popularizing applicability of the DNA of plasmids is promoted well.

Description

technical field [0001] The invention belongs to the technical field of plasmid extraction, and in particular relates to a method for extracting Escherichia coli plasmid DNA. Background technique [0002] Plasmids have the ability to replicate autonomously, enabling them to maintain a constant copy number in progeny cells and express the genetic information they carry. Bacterial plasmids are commonly used vectors in recombinant DNA technology. A vector is a tool for delivering a useful foreign gene into recipient cells for proliferation and expression through genetic engineering. A certain target gene fragment is recombined into a plasmid to form a recombinant gene or a recombinant. Then the recombinant is transformed into the recipient cell (Escherichia coli) through microbiological transformation technology, so that the target gene in the recombinant can be propagated or expressed in the recipient bacteria, thereby changing the original traits or characteristics of the ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/10C12R1/19
CPCC12N1/20C12N13/00C12N15/1003
Inventor 赵建阳王世凯韦小艳
Owner 安徽欣伯玉生物科技有限公司
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