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

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

Inactive Publication Date: 2009-07-29
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 TSB 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 used for 1.5% agarose electrophoresis detection, and the Extracted total RNA was digested with RNase-free DNaseI to prevent trace genomic DNA contamination. 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

[0037] The first ...

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Abstract

A method for constructing a cDNA library of vibrio alginolyticus poly(A) mRNA, using vibrio alginolyticus as a material, extracting total RNA, reverse transcription, and applying RD-PCR technology to amplify the double-stranded DNA obtained by reverse transcription , molecular cloning and ESTs bioinformatics methods to study the bacteria. This method has the characteristics of high quality, low repeatability of the screened gene fragments, high efficiency of screening gene fragments, and strong purpose. The library uses RD-PCR technology to amplify the gene fragments to be cloned. Since the size of the fragments after digestion is relatively uniform, it can effectively avoid the preferential amplification of short fragments by PCR and prevent the cDNA library constructed from being biased. , which is superior to the general PCR-mediated cDNA library construction method in this regard. The library can be used as the basis for in-depth research on the virulence factors and pathogenic mechanism of Vibrio alginolyticus; it can provide new ideas for the development of new genetic engineering vaccines.

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 Patents(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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