Method of simultaneously performing enzyme-cut and link up co-system based on CcdB lethal gene and SmaI restriction enzyme cutting site

A technology of enzyme cutting sites and genes, applied in the fields of biochemistry and molecular biology, to achieve the effect of speeding up the experiment progress, broad application prospects, and reducing experiment costs

Inactive Publication Date: 2017-06-30
HAINAN UNIVERSITY
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Problems solved by technology

CcdB exerts a lethal function by blocking the activity of the bacterial gyrase-DNA complex, leading to the termination of DNA replication, thereby affecting the expression of genes

Method used

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  • Method of simultaneously performing enzyme-cut and link up co-system based on CcdB lethal gene and SmaI restriction enzyme cutting site
  • Method of simultaneously performing enzyme-cut and link up co-system based on CcdB lethal gene and SmaI restriction enzyme cutting site
  • Method of simultaneously performing enzyme-cut and link up co-system based on CcdB lethal gene and SmaI restriction enzyme cutting site

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Embodiment 1

[0024] 1 Materials and methods

[0025] 1.1 Materials, reagents and strains

[0026] The Reading Frame CassetteB plasmid containing the ccdB lethal gene (its Genbank accession number is EU496090.1) was purchased from Invitrogen, the restriction enzymes EcoRI, HindIII, and SmaI were purchased from NEB, and the high-fidelity DNA polymerase Phusion-HF, T4 DNA The ligase was purchased from Thermo Fisher Scientific, and the backbone vector was pUC18. Escherichia coli strains E.coli DH 5α and DB 3.1 were preserved by our laboratory. Primers were synthesized by Huada Biological Company.

[0027] 1.2 Method

[0028] 1.2.1 Amplification of ccdB gene

[0029] Using Reading Frame Cassette B as a template, use Phusion DNA polymerase and the primers ccdB-F and ccdB-R in Table 1 to amplify the ccdB lethal fragment. The amplification program is: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 56°C for 30s , 72°C extension for 15s, 30 cycles, the final extens...

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Abstract

The invention discloses a method of simultaneously performing enzyme-cut and link up co-system based on a CcdB lethal gene and a SmaI restriction enzyme cutting site. The method comprises the following steps of: inserting the ccdB gene between EcoRI and HindIII sites of a carrier pUC18 to obtain a pU18-ccdB plasmid; and then mixing the pU18-ccdB plasmid, a DNA fragment, a restriction enzyme SmaI, T4 ligase, T4 ligase buffer and water to form a reaction system for reaction so as to obtain a link product. By taking a common pUC18 carrier as a framework carrier, a quick, efficient and low background cloning method is constructed by combining a screening mechanism of the ccdB lethal gene and the SmaI restriction enzyme cutting site contained in the CcdB lethal gene without steps of performing double digestion on a ccdB recombinant carrier and a PCR product and recovering glue and the like, and the PCR product can be directly linked to a cloning carrier, so that the method is high in efficiency; and the whole time is controlled within 20 minutes, so that the experimental cost is greatly lowered, and the experimental progress is accelerated. The cloning system has very wide application prospects.

Description

technical field [0001] The invention belongs to the field of biochemistry and molecular biology, and in particular relates to a method for simultaneously carrying out enzyme-cleavage ligation system based on CcdB lethal gene and SmaI enzyme-cleavage site. Background technique [0002] Since the advent of gene cloning technology in the early 1970s, molecular cloning has become one of the most important and basic experimental operations in modern molecular biology laboratories. The target gene fragment obtained by in vitro PCR amplification reaction often needs to be connected to the cloning vector first, which provides convenience for subsequent molecular biological operations such as sequence determination, protein expression, subcellular localization, and transgenic vector construction. Compared with directly ligating the PCR fragments into the target vector after enzyme digestion, it is simple, fast and efficient to first ligate the PCR fragments into commercial T-A clonin...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/10
CPCC12N15/10C12N15/70
Inventor 牛晓磊许文茸刘秦冯世鹏何朝族
Owner HAINAN UNIVERSITY
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