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Extraction method of bacterial RNA (Ribonucleic Acid)

An extraction method and bacterial technology, applied in the fields of molecular biology and medicine, can solve the problems of affecting use, low efficiency, low total RNA content, etc., and achieve the effects of improving extraction efficiency, simple and fast operation, and increasing concentration

Inactive Publication Date: 2017-12-12
成都晟博源生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Although the above-mentioned patent documents disclose some bacterial RNA extraction reagents, which can meet certain needs, there are still certain defects in these bacterial RNA extraction reagents: in the genomic DNA and total RNA extracted by the above method, the genomic DNA and total RNA The total RNA content is about half, and the total RNA content is low, indicating that the extraction efficiency of the above-mentioned extractant in Gram-positive bacteria total RNA is low, which affects the use

Method used

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  • Extraction method of bacterial RNA (Ribonucleic Acid)

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Embodiment 1

[0028] A method for extracting bacterial RNA, comprising the following steps:

[0029] (1) collecting the thalline, and placing the thalline in a centrifuge tube;

[0030] The method of collecting the bacteria is as follows: firstly, the bacteria liquid is placed in a centrifuge tube and centrifuged, and then all the culture medium supernatant is removed.

[0031] (2) Add 50 μl of EB buffer solution containing lysozyme to the centrifuge tube to completely resuspend the bacteria;

[0032] (3) Add 200 μl buffer A to the centrifuge tube, and mix by vortexing; if insoluble precipitate appears, centrifuge it, and transfer the supernatant obtained after centrifugation to another centrifuge tube;

[0033] Among them, buffer A is a bacterial cell wall breaking reagent, including guanidine isothiocyanate, guanidine hydrochloride and β-mercaptoethanol, wherein the concentration of guanidine isothiocyanate is 1M, the concentration of guanidine hydrochloride is 1M, and the content of β-m...

Embodiment 2

[0046] A method for extracting bacterial RNA, comprising the following steps:

[0047] (1) collecting the thalline, and placing the thalline in a centrifuge tube;

[0048] The method of collecting the bacteria is as follows: firstly, the bacteria liquid is placed in a centrifuge tube and centrifuged, and then all the culture medium supernatant is removed.

[0049] (2) Add 200 μl of EB buffer containing lysozyme to the centrifuge tube to completely resuspend the bacteria;

[0050] (3) Add 600 μl buffer A to the centrifuge tube, and mix by vortexing; if insoluble precipitate appears, centrifuge it, and transfer the supernatant obtained after centrifugation to another centrifuge tube;

[0051] Among them, buffer A is a bacterial cell wall breaking reagent, including guanidine isothiocyanate, guanidine hydrochloride and β-mercaptoethanol, wherein the concentration of guanidine isothiocyanate is 5M, the concentration of guanidine hydrochloride is 5M, and the content of β-mercaptoe...

Embodiment 3

[0064] A method for extracting bacterial RNA, comprising the following steps:

[0065] (1) collecting the thalline, and placing the thalline in a centrifuge tube;

[0066] The method of collecting the bacteria is as follows: firstly, the bacteria liquid is placed in a centrifuge tube and centrifuged, and then all the culture medium supernatant is removed.

[0067] (2) Add 145 μl of EB buffer solution containing lysozyme to the centrifuge tube to completely resuspend the bacteria;

[0068] (3) Add 450 μl buffer A to the centrifuge tube, and mix by vortexing; if insoluble precipitate appears, centrifuge it, and transfer the supernatant obtained after centrifugation to another centrifuge tube;

[0069] Among them, buffer A is a bacterial cell wall breaking reagent, including guanidine isothiocyanate, guanidine hydrochloride and β-mercaptoethanol, wherein the concentration of guanidine isothiocyanate is 2M, the concentration of guanidine hydrochloride is 3M, and the content of β-...

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Abstract

The invention discloses an extraction method of bacterial RNA (Ribonucleic Acid). The extraction method comprises the following steps: firstly, collecting thallus and then putting the thallus into a centrifuge tube; secondly, adding 50 to 200mu l of EB buffer solution containing lysozyme into the centrifuge tube for thoroughly resuspending the thallus; thirdly, adding 200 to 600mu l of buffer A into the centrifuge tube and then uniformly mixing by vortex oscillation; if the insoluble precipitate occurs, centrifuging the insoluble precipitate, and transferring supernatant obtained by centrifuging into another centrifugal tube; fourthly, adding 200 to 600mu l of buffer B into the centrifuge tube, uniformly mixing, then adding a magnetic bead solution, oscillating, uniformly mixing and the like. According to the extraction method of the bacterial RNA, provided by the invention, the thallus can be quickly cracked, and further the RNA is released; in addition, the magnetic bead is combined with the advantages of nucleic acid, so that the extraction concentration is increased, the operation is simple and quick, and the extraction efficiency is greatly improved.

Description

technical field [0001] The invention relates to the fields of molecular biology and medicine, in particular to a method for extracting bacterial RNA. Background technique [0002] RNA is a messenger or template that exists in biological cells and is used to transfer genetic information from DNA to functional proteins. In recent years, with the progress of bacterial genetic engineering, we have ushered in the post-genome era of bacteria. It has become a research hotspot to explain the function of genes and the regulatory mechanism of gene expression through the study of gene expression profiling. RNA extraction technology is not only an important part of molecular biology technology, but also an important basis for functional genomics research technology. With the advent of the post-genome era of bacteria, seeking an effective reagent and method for extracting bacterial RNA has become an urgent problem to be solved. [0003] The patent document with the application number Z...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 贾雪荣张文宝王亚周
Owner 成都晟博源生物工程有限公司
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