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A kind of construction method of small RNA cDNA library

A DNA molecule and library technology, which is applied in the field of SmallRNA cDNA library construction, can solve the problems of paralysis of the sequencer, expensive equipment and high consumption, and achieves the effect of simple and quick method, increasing the content of target products and reducing non-specific products.

Active Publication Date: 2018-03-02
SUZHOU GENEPHARMA
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Problems solved by technology

[0002] Biotechnology and life science will become an important driving force for the new scientific and technological revolution in the 21st century. Many countries, including my country, have listed it as a key research field for priority development. Gene sequencing technology, as the core common technology for the development of the biological industry, It has become the commanding heights of science and technology that developed countries are competing to seize, but the related technologies are monopolized by a few European and American companies and the equipment is expensive
[0003] Gene sequencing requires gene sequencers and sequencing reagents, and the production of gene sequencers and kits involves many technologies such as organic chemical synthesis, genetic modification, catalytic enzyme synthesis, and sequencing. The process is complicated and the quality requirements are high. At present, only There are three American manufacturers, and the cost of reagents for one instrument is more than 10 million yuan a year
Due to high consumption, many high-priced sequencers in my country are idle and paralyzed

Method used

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  • A kind of construction method of small RNA cDNA library
  • A kind of construction method of small RNA cDNA library
  • A kind of construction method of small RNA cDNA library

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Embodiment 1

[0087] Embodiment 1, the construction of Small RNA cDNA library

[0088] The roadmap for the construction of the Small RNA cDNA library is as follows: figure 1 shown.

[0089] 1. Isolation of Small RNA

[0090] (1) Extract total RNA from 293T cells, take 10 μg and dilute to 200 μL with nuclease-free water.

[0091] (2) Transfer the total RNA to an ultrafiltration column, and centrifuge at 12,000 rpm for 15 minutes to obtain a filtrate.

[0092] (3) Transfer the filtrate to a 1.5mL tube, add 1 / 10 volume of 3M NaAc and 3 volumes of absolute ethanol, and freeze at -20°C for 30min.

[0093] (4) Centrifuge at 12,000 rpm at 4°C for 30 minutes, remove the supernatant, and obtain a precipitate.

[0094] (5) Add 1 mL of 80% by volume ethanol aqueous solution to the precipitate, centrifuge at 12,000 rpm at 4°C for 5 min, remove the supernatant, and air-dry to obtain a precipitate.

[0095] (6) Repeat step (5) once, add 10 μL of nuclease-free water to fully dissolve the precipitate,...

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Abstract

The invention discloses a method for constructing a Small RNA cDNA library. A group of DNA molecules disclosed by the present invention, the group of molecules is composed of four molecules as shown in (1)-(4): (1) the DNA molecule shown in SEQ ID No.1; (2) SEQ ID No. The DNA molecule shown in 2; (3) the DNA molecule shown in SEQ ID No.3; (4) the DNA molecule shown in SEQ ID No.4. The method for rapidly separating Small RNA disclosed by the invention solves the problems of long time, complicated operation, high requirements and high toxicity of the current separation of Small RNA by acrylamide gel electrophoresis. Moreover, the method for constructing a Small RNA cDNA library disclosed in the present invention reduces non-specific products and increases the content of target products, and the method is simpler and faster than traditional methods.

Description

technical field [0001] The invention relates to a method for constructing a Small RNA cDNA library. Background technique [0002] Biotechnology and life science will become an important driving force for the new scientific and technological revolution in the 21st century. Many countries, including my country, have listed it as a key research field for priority development. Gene sequencing technology is the core common technology for the development of the biological industry. It has become the commanding heights of science and technology that developed countries are competing to seize, but the related technologies are monopolized by a few European and American companies and the equipment is expensive. [0003] Gene sequencing requires gene sequencers and sequencing reagents, and the production of gene sequencers and kits involves many technologies such as organic chemical synthesis, genetic modification, catalytic enzyme synthesis, and sequencing. The process is complicated a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/6806
Inventor 郭良让孙少华苗茹
Owner SUZHOU GENEPHARMA
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