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Cloning vector and preparation and application thereof

A cloning vector and vector technology, applied in the field of genetic engineering, can solve problems such as increased experimental cost, high price, and inability to grow

Inactive Publication Date: 2016-03-16
生工生物工程(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, the efficiency of TA cloning depends on the quality of T vectors. At present, only a few biotechnology companies can provide high-quality T vectors and the price is high. Generally, laboratories need to purchase T vectors, which increases the cost of experiments.
[0009] Common Escherichia coli strains cloned with ccdB gene (such as DH5α, Top10, etc.) cannot grow due to the toxicity of CcdB protein

Method used

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  • Cloning vector and preparation and application thereof
  • Cloning vector and preparation and application thereof
  • Cloning vector and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of vector pUC57-ccdB

[0036] 1. Synthesis of 1ccdB gene

[0037] The gene was synthesized by overlap extension PCR, firstly, ccdB-1, ccdB-2, ccdB-3, ccdB-4 were used as the first set of primers, and ccdB-3, ccdB-4, ccdB-5, ccdB-6 were used as the second set Primers, ccdB-5, ccdB-6, ccdB-7, and ccdB-8 are the third set of primers PCR to obtain three small ccdB gene fragments, and then use the mixture of these three fragments as a template to use primers ccdB-1 and ccdB -8PCR to obtain the complete ccdB gene. The obtained gene was detected by 1.5% agarose gel electrophoresis, and the target fragment was recovered with the "SanPrep Column Gel Recovery Kit" provided by Sangon (Bioengineering) Co., Ltd. to obtain the ccdB gene synthesis product.

[0038] The primer sequence of synthetic ccdB gene is as follows:

[0039] ccdB-1 (SEQ ID NO: 3):

[0040] 5'-cggcaattc catatg caatttaaggtgtatacctacaaacgtgaatctcg-3' (with NdeI)

[0041] ccdB-2 (SEQ I...

Embodiment 2

[0058] Example 2: Application of vector pUC57-ccdB

[0059] The target fragment amplified by high-fidelity enzyme PCR was recovered and mixed with the carrier pUC57-ccdB, added with SmaI restriction endonuclease, T4 DNA ligase, etc., and digested and ligated overnight at 22°C. Three genes with different lengths of about 800bp (restriction enzyme SphI gene), 1200bp (restriction enzyme XmnI gene), and 2500bp (TaqDNA polymerizing gene) are used as examples for illustration.

[0060] 2.1 Construction of pUC57-ccdB-SphI

[0061] The SphI gene was obtained by PCR with Pfu enzyme, and after detection by 1% agarose gel electrophoresis, the PCR product was purified by a gel recovery kit, and the concentration was determined to be 65 ng / μL by a microplate reader after purification.

[0062] The enzyme digestion ligation system is as follows:

[0063]

[0064] Ligation overnight at 22°C. Take 10 μL of the ligation product to transform Escherichia coli DH5α competent cells, pick 10 ...

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Abstract

The invention relates to a cloning vector and preparation and application thereof and discloses a cloning vector pUC57-ccdB which is a modified vector with ccdB genes inserted at multiple cloning sites of a pUC57 vector, wherein the ccdB genes have blunt-end restriction enzyme digested recognition sites. By means of the lethal effect of CcdB protein on escherichia coli not containing F plasmids, the ccdB genes having the restriction enzyme Sma I digested sites are inserted on the pUC57 plasmids through the molecular biological technology to obtain the vector pUC57-ccdB. Blunt ends are generated through Sma I digestion and are connected with the genes needing to be cloned so that insertion of the genes needing to be cloned can be achieved. Meanwhile, bacterial colonies with empty vectors are avoided. The cloning vector plays an important role in the fields of molecular biology and genetic engineering.

Description

technical field [0001] The invention relates to a cloning carrier and its preparation and application, belonging to the field of genetic engineering. Background technique [0002] The invention of PCR technology is a major breakthrough in the fields of molecular biology and genetic engineering. After the birth of PCR technology, the technology of cloning PCR products into vectors (usually plasmids) has also been developed. At present, a variety of methods for constructing clones have been developed, such as restriction endonuclease digestion and ligation (restrictionenzymedigestionandligation), ligation-independent cloning (LIC), in vivo ligation (invivoligation) and site-specific recombination system (site -specific recombination systems), etc. However, these methods require the use of restriction enzymes to treat PCR products and vectors, or require special strains or some special enzymes, so the operation is complicated and cumbersome, and it is difficult to perform hig...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66
CPCC12N15/63C12N15/66C12N2800/101
Inventor 傅向阳
Owner 生工生物工程(上海)股份有限公司
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