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Rapid and efficient DNA recombination method and kit

A DNA recombination and kit technology, applied in the field of genetic engineering, can solve the problems of easy fragmentation, difficult operation of macromolecular DNA, ligation, and reduced transformation efficiency, and achieve the effect of high efficiency and simple operation.

Inactive Publication Date: 2010-12-01
万俊松
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Problems solved by technology

In addition, the in vitro manipulation of macromolecular DNA is difficult and easy to break, and the efficiency of connection and transformation also decreases with the increase of the length of DNA fragments.

Method used

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  • Rapid and efficient DNA recombination method and kit
  • Rapid and efficient DNA recombination method and kit

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Embodiment Construction

[0019] In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the present invention will be further described below in conjunction with specific illustrations.

[0020] A method and kit for fast and efficient DNA recombination, first prepare the DNA recombination kit:

[0021] 1. Extract the total DNA of Photobacteria

[0022] Inoculate Photobacteria to LB slant, culture at 30°C for 6-8 hours to activate, then place a loop in 50ml LB liquid medium, culture at 30°C, 200r / min shaker overnight. The cells were collected by centrifugation and washed once with TES solution (10mM Tris-HCl, pH8.0, 1mM EDTA, 100mM NaCl). Centrifuge, resuspend the pellet in TE1 (25% sucrose, 25mM Tris-HCl, pH8.0, 25mM EDTA), add sodium dodecyl sulfate (SDS) to a final concentration of 2%, mix well to lyse the cells, add 1M NaCl solution, incubate at 37°C for 5min. Centrifuge, get the supernatant, extract with equal volume of ph...

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Abstract

The invention relates to a rapid and efficient DNA recombination method and a kit. The method comprises the following steps: extracting the total DNA of Photorhabdus; taking the extracted total DNA as a template to carry out PCR amplification of a plu2935 gene and a plu2936gene; cloning the plu2935 gene and the plu2936 gene into a pET32a vector, and constructing a recombinase expression vector pET-plu; introducing the pET-plu into BL21(DE3) to obtain engineering bacteria BL21-REC capable of expressing recombinases Plu2935 and Plu2936; utilizing IPTG to induce the BL21-REC, expressing the recombinase of Plu2935 and Plu2936, and preparing into a competent cell; and introducing two DNA fragments comprising homologous sequences into the BL21-REC competent cell, and screening on a corresponding resistant panel to obtain the transformant of the recombinant DNA.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method and a kit for fast and efficient DNA recombination capable of performing deletion, mutation and insertion of exogenous DNA fragments on target DNA. Background technique [0002] Since the advent of recombinant DNA technology in the early 1970s, after nearly 40 years of development, both in the field of basic theoretical research and in practical production applications, amazing achievements have been made. Recombinant DNA technology has not only brought about unprecedented profound changes in the research of the entire life sciences, but also strongly promoted the development of medical science research. Considerable achievements have been made in many sciences such as research. [0003] However, it is difficult to modify macromolecular DNA such as BAC library by traditional DNA recombination technology using restriction endonuclease and ligase as the main tools. In ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/10
Inventor 万俊松
Owner 万俊松
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