A method for constructing t vector

A carrier and precursor technology, applied in the field of genetic engineering, can solve the problems of many quality control links, low quality of the T carrier, and cumbersome production process, reducing operation steps and control links, high enzyme digestion yield, and self-cyclization. low rate effect

Inactive Publication Date: 2011-12-21
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are two defects in this method: one is that the addition of T to the 3′ end of the linear plasmid may not be complete under the action of DNA polymerase, resulting in a high self-circularization rate during the cloning process and a high false positive rate; EcoRV enzyme digestion, DNA polymerase plus T, electrophoresis gel recovery of DNA fragments and other steps require many steps of quality control, and there are many factors that affect the cloning efficiency of T vectors, and the quality of T vectors is low
At the same time, due to the cumbersome production process and high cost of T carriers, the price of commercial T carriers will eventually be higher

Method used

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  • A method for constructing t vector
  • A method for constructing t vector
  • A method for constructing t vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Amplification of the resistance gene fragment containing AhdI site

[0042] Using the pEGFP-C1 plasmid as a template, use the upstream primer with the nucleotide sequence shown in SEQ ID No: 2 and the downstream primer with the nucleotide sequence shown in SEQ ID No: 3 to carry out PCR amplification to obtain the The kanamycin resistance gene fragment at the AhdI site.

[0043] The PCR amplification reaction system is:

[0044]

[0045] Add sterilized distilled water to make up the total amount of the system to 20 μL.

[0046] The PCR reaction procedure is:

[0047]

[0048] The resulting PCR product was purified and recovered, and 5 μL of 1% agarose gel electrophoresis was used for detection. The results are shown in figure 1

Embodiment 2

[0049] Example 2: Preparation of T carrier precursor

[0050] The PCR product recovered in Example 1 and the pUC-19 plasmid were double-digested with BamHI and HindIII respectively (the endonuclease and corresponding buffer used were Takara products), and the reaction system was as follows:

[0051] Add the following ingredients to Tube A:

[0052]

[0053] Add sterilized distilled water to make up the total amount of the system to 30 μL.

[0054] Add the following ingredients to tube B:

[0055]

[0056] Add sterilized distilled water to make up the total amount of the system to 30 μL.

[0057] Put tubes A and B in a constant temperature water bath at 37°C for 6-8 hours for digestion and digestion, and then perform gel electrophoresis and DNA gel recovery for the digested products. The recovered fragments were ligated overnight at 16°C under the action of T4 DNA ligase (8-12h).

[0058] Connection reaction system:

[0059]

[0060]

[0061] Add sterilized dis...

Embodiment 3

[0063] Embodiment 3: Preparation of T carrier

[0064] The T vector precursor obtained in Example 2 was digested with the specific restriction endonuclease AhdI in the following manner, and the linear vector with a size of about 2000 bp was recovered by electrophoresis, which was the T vector.

[0065] Enzyme digestion reaction system:

[0066]

[0067] Add sterilized distilled water to make up the total amount of the system to 30 μL.

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Abstract

The invention relates to the technical field of genetic engineering, and discloses a method for constructing a T vector, in order to directionally clone a resistance gene containing a specific restriction endonuclease recognition sequence at both ends between two multiple cloning sites of the starting vector, Screen positive clones with antibiotics corresponding to the above-mentioned resistance genes to obtain T vector precursors; then digest the obtained T vector precursors with the specific restriction endonuclease, recover large fragments, and obtain T vectors; wherein, the specific restriction endonuclease The endonuclease whose recognition sequence is digested can be a restriction endonuclease whose protruding terminal site can be T, and the resistance gene is different from the resistance gene contained in the starting vector. The method for constructing the T vector in the present invention is simple and efficient, and the quality of the T vector is controlled by using the introduced resistance gene fragment during the preparation process. The T vector obtained by this method not only retains the advantages of the starting vector, but also has the characteristics of stable quality, low self-cyclization rate, and high cloning effect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing a T vector. Background technique [0002] Gene cloning technology is a basic technology of modern genetic engineering research and a prerequisite for studying gene structure and function. Using T vectors for gene cloning is one of the most simple and convenient methods. The T vector is a cloning vector for polymerase chain reaction products, and after linearization, there is one more deoxythymidylic acid (T) at the 3' end of both sides. [0003] When Taq DNA polymerase is used to amplify the gene by PCR, a deoxyadenosine (A) base will be added to the 3' end of the PCR product, which can pair with the protruding T base at the 3' end of the T carrier. Under the action of DNA ligase, a closed circular molecule is formed, and then transformed into Escherichia coli competent cells, cultured and grown into clonal colonies. Colony PCR and restrict...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/63
Inventor 周江宁方辉赵君
Owner UNIV OF SCI & TECH OF CHINA
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