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Method for constructing cDNA (complementary DNA) library of soil microbe

A technology of soil microorganisms and construction methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of cumbersome operation process, easy RNA degradation, and complicated operation steps, so as to achieve no operation steps and convenient method , the effect of rich sources of information

Inactive Publication Date: 2013-04-17
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method is not only time-consuming, but also prone to RNA degradation due to the cumbersome operation process, which leads to experimental failure.
[0004] Therefore, it is necessary to develop a fast, simple and efficient method for constructing soil microbial cDNA library to solve the problem of complex routine operation steps and easy RNA degradation. This method is necessary for the study of functional diversity of soil microorganisms.

Method used

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  • Method for constructing cDNA (complementary DNA) library of soil microbe
  • Method for constructing cDNA (complementary DNA) library of soil microbe
  • Method for constructing cDNA (complementary DNA) library of soil microbe

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Experimental program
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Embodiment 1

[0032] The construction method of soil microorganism cDNA library of the present invention, concrete steps are as follows:

[0033] (1) Raw material: fresh rice rhizosphere soil from Fuzhou, Fujian (teaching farm of Fujian Agriculture and Forestry University);

[0034] (2) Extraction of total RNA of soil microorganisms: the extraction of total RNA of soil microorganisms was carried out with reference to the method in the patent specification of Fang Changxun et al. (A method for extracting genomic DNA and total RNA of soil microorganisms, application number: 201010548527.X). The obtained soil microbial genomic DNA and total RNA were extracted according to this method, and the total RNA was obtained after removing the genomic DNA by using DNase (DNAase I, TaKaRa Biotechnology, Dalian) at 37°C for 10 minutes, and the total RNA was quickly extracted with RNAPure high-purity total RNA The kit (Beijing Biotech Co., Ltd.) and MicroSpin S-400 HR spin column (GE Healthcare, Little Cha...

Embodiment 2

[0041] (1) Take fresh sugarcane rhizosphere soil from Fuzhou, Fujian (Fujian Agriculture and Forestry University sugarcane garden); the extraction of total RNA of soil microorganisms refers to Fang Changxun et al. (A method for extracting genomic DNA and total RNA of soil microorganisms, application number: 201010548527.X) by the method of the patent specification. The obtained soil microbial genomic DNA and total RNA were extracted according to this method, and the total RNA was obtained after removing the genomic DNA by using DNase (DNAase I, TaKaRa Biotechnology, Dalian) at 37°C for 10 minutes, and the total RNA was quickly extracted with RNAPure high-purity total RNA The kit (Beijing Biotech Company) was purified by column using MicroSpin S-400 HR spin column (GE Healthcare, Little Chalfont, UK).

[0042] (3) Reverse transcription of total RNA: The total RNA obtained in (2) above was reverse-transcribed using PrimeScript RT reagent Kit (TaKaRa Biotechnology, Dalian). The ...

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Abstract

The invention provides a method for constructing a cDNA (complementary DNA) library of a soil microbe. The method comprises the following steps of: reversely transcribing a total RNA into a single-chain cDNA at first; performing dephosphorylation on a 5' end of the single-chain cDNA, and then connecting the 5' end of the single-chain cDNA with a phosphorylation-modified joint 1 of the 5' end; performing phosphorylation reaction on the cDNA of which the 5' end is connected with the joint; connecting a 3' end of the cDNA which is subjected to the phosphorylation reaction with a joint 2 to obtain a cDNA of which the 5' end and the 3' end are connected with a joint respectively; amplifying the cDNA by adopting oligonucleotide which is complementary with the sequences of the joint parts as a primer so as to obtain a double-chain cDNA of the soil microbe; and connecting the double-chain cDNA with a cloning vector pMD-18T, and then transforming Escherichia coli competent cells to finally obtain the cDNA library of the soil microbe. The cDNA library of the soil microbe constructed by the method has a larger storage capacity, can be used for screening interested target genes, and analyzinginteraction of subsequent genes and proteins.

Description

technical field [0001] The invention relates to a method for constructing a soil microbial cDNA library, belonging to the field of biotechnology. Background technique [0002] With the rapid development of biology and information technology, excavating, cloning new genes, and studying the functions of new genes have become an important task in functional genome research. The construction of cDNA library is one of the basic means of functional genomics research. It has special advantages in the study of the expression state of the genome in a specific type of cell and the function identification of expressed genes, so that it can be used in individual development, cell differentiation, cell It has wider application value in the study of life phenomena such as cycle regulation, cell aging and death regulation, and is the most commonly used gene library in research work. [0003] As an important part of the soil ecosystem, soil microorganisms play an important role in biogeoch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C40B40/08C12N1/21C12N15/70C12N15/10C12R1/19
Inventor 方长旬林文雄黄力坤许铁城林瑞余
Owner FUJIAN AGRI & FORESTRY UNIV