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Construction method for large fragment DNA clone carrier

A technology of DNA cloning and construction methods, which is applied in the direction of recombinant DNA technology, DNA/RNA fragments, and the use of vectors to introduce foreign genetic materials. Accidental mutation, improving capacity and stability of exogenous gene, effect of small molecular weight

Inactive Publication Date: 2008-06-11
SOUTH CHINA AGRI UNIV
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Problems solved by technology

In addition to pBR322, pUC series, pGEM series, phage, and cosmids, the vectors used now include yeast artificial chromosomes, bacterial artificial chromosomes, etc. Although these vectors have been widely used, due to the limitation of the foreign gene cloning window area Endonuclease recognition sites are not easy to replace, which brings many difficulties to the cloning of some specific DNA fragments, especially larger fragments

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  • Construction method for large fragment DNA clone carrier

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Embodiment 1

[0027] 1. Materials

[0028] 1. Plasmid and bacterial strain: pBR322 cloning vector (accession No.: J01749), template for backbone amplification, purchased from Takara Company. DH5 α competent cells were preserved by the Department of Poultry Diseases, South China Agricultural University.

[0029] 2. Enzymes and other reagents: T4 Polynucleotide Kinase, PrimeSTAR  HS DNA Polymerase and DNA Ligation Kit were purchased from Takara Company. Gel Extraction Kit and Plasmid mini Kit (200) were purchased from OMEGA Company.

[0030] 3. Primers: According to the pBR322 cloning vector sequence, design reference primers (VF, VR) for amplifying the 2471-4180 region containing Amp and ori, synthesized by Takara Company, the sequence is:

[0031] VF: 5'-cggaacatgtgagcaaaa-3'

[0032] VR: 5'-cttcaataatattgaa-3'

[0033] Specific steps:

[0034] See attached drawing 1 for connection diagram, where A: vector backbone, including AmpR gene of pBR322 and ori origin of replication; B, C: ...

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Abstract

The invention discloses a method for simply and quickly constructing cloning carrier, which is a LJY method for short, and has 4 cloning carriers which are constructed through the method. A AmpR gene of pBR322 and ori copying initial point are used as the skeleton of the invention, through the unique primer designing method and blunt end connection, a required restriction endonuclease recognition site is simply and quickly led into new constructed cloning carrier plasmid, and the exogenous gene capacity and the stability of the carrier are effectively enhanced, to avoid the often appearing problem that a target gene is suddenly changed when large fragment genes are cloned.

Description

technical field [0001] The invention relates to biological cloning technology, in particular to a method for simply and quickly constructing a large-segment DNA cloning vector. Background technique [0002] Cloning fragments of DNA is routine work in molecular biology. Since Cohen et al. (1973) introduced the plasmid pSC101 as a cloning vector, a large number of vector plasmids with excellent performance have been successfully constructed and commercialized. In addition to pBR322, pUC series, pGEM series, phage, and cosmids, the vectors used now include yeast artificial chromosomes, bacterial artificial chromosomes, etc. Although these vectors have been widely used, due to the limitation of the foreign gene cloning window area The recognition site of the endonuclease is not easy to replace, which brings many difficulties to the cloning of some specific DNA fragments, especially larger fragments. Contents of the invention [0003] The purpose of the present invention is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/11C12N15/66
Inventor 廖明江经纬郁宏伟周燕芬
Owner SOUTH CHINA AGRI UNIV
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