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Process of constructing poly (A) mRNA cDNA library of vibrio alginolyticus

A technology for Vibrio alginolyticus and library construction, which is applied in chemical libraries, biochemical equipment and methods, libraries, etc., and can solve problems such as fewer tails, redundancy, and difficulty in mRNA purification

Inactive Publication Date: 2007-08-22
GUANGDONG OCEAN UNIVERSITY
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Problems solved by technology

This is because the metabolism of bacterial mRNA is fast and efficient, the half-life is only a few minutes, and the poly(A) tail at the 3′ end is less, short and unstable, which makes the purification of mRNA and reverse transcription with oligo(dT) more difficult. Eukaryotic cDNA library construction method, even if the cloning is successful, there will be a lot of redundant and repeated information in the cDNA library, and the obtained multiple clones of different sizes may all come from the mRNA of the same gene in different states

Method used

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Embodiment 1

[0033] (1) Template preparation

[0034] ①Take the preserved Vibrio alginolyticus strain, culture it at 28°C for 24 hours after streaking on the plate, pick a single colony and transfer it to 100ml liquid TSA medium, and culture it with shaking at 28°C for 8 hours. The culture time of the bacteria should not be too long. -10 hours is better, otherwise the RNA extraction effect is not good and it is easy to degrade. Test A 600 about 0.6;

[0035] ②The preparation of total RNA was carried out using the EZ spin column total RNA lsolation Kit (purchased from Shanghai Sangon Bioengineering Co., Ltd.), quantified with a BIO-TEK microplate reader, and 2 μL RNA was subjected to 1.5% agarose electrophoresis detection, and the extracted The total RNA was digested with RNase-free DNase I to prevent contamination from trace genomic DNA. The mRNA was purified using Omega Biotek's E.Z.N.A mRNA Enrichment Kit, and operated according to the instructions;

[0036] (2) cDNA synthesis

[00...

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Abstract

The process of constructing poly(A) mRNA cDNA library of Vibro alginolyticus performs research on Vibro alginolyticus through extracting total RNA, reverse transcription, RD-PCR amplification and reverse transcription to obtain double strand DNA, molecular cloning and ESTs biological informatic step, etc. The process has the features of high quality, low repeatability of the screened gene segment, high efficiency of screening gene segment, etc. The process is superior to common PCR mediated cDNA library constructing process, and the constructed library may be used in deep research of virulence factor and pathogenic mechanism of Vibro alginolyticus and in developing new type of genetic engineering vaccine.

Description

Technical field: [0001] The invention belongs to the fields of microbiology, biochemistry, molecular biology and bioinformatics, and in particular relates to a method for constructing a cDNA library of Vibrio alginolyticus poly(A) mRNA. Background technique: [0002] Vibrio alginolyticus (Vibrio alginolyticus) is a Gram-negative halophilic bacterium that widely exists in seawater and seafood all over the world. Important pathogens causing food poisoning and acute diarrhea in humans. At present, most scholars' research on this bacterium mainly focuses on the outer membrane protein and the idiotype monoclonal antibody against Vibrio alginolyticus. However, the research on the library construction, functional genes and virulence factors of Vibrio alginolyticus is still lacking. [0003] Since the first cDNA clone came out in 1976, the cDNA library construction method has made great progress and has become the basic method of molecular biology in eukaryotes, but it has almost s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C40B50/06C12N15/31
Inventor 吴灶和王蓓简纪常鲁义善
Owner GUANGDONG OCEAN UNIVERSITY
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