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Method for constructing Small RAN cDNA library

A DNA molecule and library technology, applied in the field of Small RNA cDNA library construction, can solve the problems of high consumption, paralysis of the sequencer, complicated process, etc., and achieve the effect of increasing the content of the target product, reducing the non-specific product, and the method is simple and fast

Active Publication Date: 2015-05-20
SUZHOU GENEPHARMA
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Problems solved by technology

[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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  • Method for constructing Small RAN cDNA library
  • Method for constructing Small RAN cDNA library
  • Method for constructing Small RAN 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 RAN cDNA library. A group of DNA molecules disclosed by the invention consist of the following four kinds of molecules (1)-(4): (1) DNA molecule shown as SEQ ID No. 1; (2) DNA molecule shown as SEQ ID No. 2; (3) DNA molecule shown as SEQ ID No. 3; and (4) DNA molecule shown as SEQ ID No. 4. The Small RNA quick separation method solves the problems of long time, complicated operation, high requirement and high toxicity in acrylamide gel electrophoresis separation of Small RNA at present. Moreover, the method for constructing the Small RAN cDNA library reduces nonspecific products, improves the content of a destination product and is simple, convenient and fast compared with a traditional method.

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 Applications(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/68
Inventor 郭良让孙少华苗茹
Owner SUZHOU GENEPHARMA
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