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Kit for rapid construction of recombinant plasmid by using ccdB lethal gene

A kit and gene technology, applied in the field of molecular biology, can solve problems such as false positives, achieve low random error, reduce false positive rate, and realize the effect of automatic operation

Inactive Publication Date: 2019-10-08
成都橘井生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has the disadvantage of false positives: the PCR product still contains a part of the unamplified circular empty vector, and the circular empty vector and the linear target gene will not recombine, and the competent cells with the circular empty vector Can still form clones under antibiotic selection, thus generating false positives

Method used

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  • Kit for rapid construction of recombinant plasmid by using ccdB lethal gene
  • Kit for rapid construction of recombinant plasmid by using ccdB lethal gene
  • Kit for rapid construction of recombinant plasmid by using ccdB lethal gene

Examples

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Effect test

Embodiment 1

[0060] Embodiment 1 uses the method of the present invention to carry out the rapid clone Mqo gene multi-carrier utilizing ccdB gene reverse screening

[0061] The vectors used in this example include plasmid vectors: pFastV1-V12 are obtained by inserting a common sequence into commercial vectors. The corresponding commercial vectors are: pET28a pET29a, pET30b, pET33b, pET28aMBP, pET28aMBPC, pGEX6p-1, pGEX4T-1, pETDuet1, pCold1, pET15b, pET32a. Each vector carries a kanamycin resistance gene.

[0062] The common sequence: ctggtgccgc gcggcagcgg aggaggaatc atcatc (SEQ ID NO: 1). In this example, the HF and HR primers are amplified by matching the "common sequence".

[0063] The universal primers used to verify the sequences of the aforementioned plasmid vectors are shown in Table 1 and Table 2.

[0064] Various reagents and carriers are commercially available. Among them, MCLab DNA Polymerase Mix is ​​from Beijing Qingke Biotechnology Co., Ltd. Chengdu Branch, Utaq DNA Polym...

Embodiment 2

[0091] Embodiment 2 Kit of the present invention

[0092] The test kit of the present invention consists of the following components:

[0093] (1) Primers HF and HR are the same as HF and HR in Example 1;

[0094] (2) A circular vector with a ccdB gene expression cassette, the same as the pFastB1-B12 vector in Example 1.

[0095] In summary, the present invention can get rid of the dependence on restriction endonucleases, and does not require in vitro ligation reaction, has a low false positive rate, and efficiently and quickly constructs genes into multiple vectors.

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Abstract

The present invention relates to the field of molecular biology, and more specifically relates to a method for rapidly constructing a recombinant plasmid using a ccdB lethal gene and a kit thereof. The method combines PCR and a mechanism of homologous recombination in bacteria to get rid of the dependence on DNA digestion and ligation in vector construction; the ccdB lethal gene is introduced, andthe screening of positive clones can be simplified. The kit can be used for high-throughput carrier construction, and has the advantages of time saving, labor saving and low cost.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method and a kit for rapidly constructing a recombinant plasmid using a ccdB lethal gene. Background technique [0002] Plasmids are the basic tools of modern biological research, and have been applied to all aspects of life science research and medical health development, such as library establishment, protein expression, gene therapy, gene editing and DNA vaccines. Molecular cloning technology is the process of inserting the amplified target DNA fragment into the target plasmid vector. It is one of the core technologies of modern biomedical research. Plasmid construction has almost become the primary work of all basic biomedical research. With the promotion of the third-generation gene sequencing technology, a large amount of digital genetic information needs to be transformed into various recombinant plasmids necessary for basic biomedical research. The rapid establishment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/70
CPCC12N15/66C12N15/70
Inventor 苏丹江华卢德仁
Owner 成都橘井生物科技有限公司
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