Design and construction method of high efficiency temperature controlled positive screening cloning carrier

A cloning vector and positive screening technology, which can be used in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., and can solve the problems of cumbersome operation, time-consuming and laborious

Inactive Publication Date: 2008-05-21
SOUTHWEST UNIVERSITY
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Problems solved by technology

This kind of negative screening technology is an early technology in the era of genetic engineering. It needs to copy

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  • Design and construction method of high efficiency temperature controlled positive screening cloning carrier

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Embodiment 1

[0024] Implementation Example 1: Construction of common cloning vector pClonEZ

[0025] Taking the non-patented plasmid vector pBV220 widely used in domestic laboratories as the starting vector, it was constructed according to the following steps and methods:

[0026] 1. Using the genomic DNA of the commercially available phage Φ×174 as a template, the complete E gene coding sequence (273 bp) was amplified by PCR technique. When designing primers for amplifying the E gene, an EcoRI site was introduced into the upstream primer, a PstI site was introduced into the downstream primer, and 4 protective bases were introduced outside the restriction site. PCR amplification was carried out according to the selected high-fidelity Taq enzyme product manual and the Tm value of the primers. The size of the amplified product is about 300bp.

[0027] 2. Use EcoRI and PstI to double-digest plasmid pBV220 and the above PCR-amplified E gene fragment respectively, and tap rubber to recover th...

example 2

[0036] Example 2: Construction of T vector pEZ-T

[0037] The T vector pEZ-T for T-A cloning is constructed with the pClonEZ constructed in Example 1 as a starting vector:

[0038] 1. Use the commercially available QuikChange(R) site-directed mutagenesis kit and carry out point mutations to pClonEZ according to the method described in its product manual, and mutate the EcoR I site sequence before the E gene into the EcoR V site sequence, and the resulting plasmid is pEZ-T pre-carrier.

[0039] 2. Digest the above pEZ-T pro-vector with EcoR V, and the enzyme digestion reaction system and conditions are carried out according to the corresponding endonuclease product instructions.

[0040] 3. Purify and recover the linearized pEZ-T procarrier by conventional phenol-chloroform extraction and ethanol precipitation methods.

[0041] 4. Using the above-mentioned pEZ-T pro-vector and dTTP as substrates, use Taq DNA polymerase to carry out terminal T addition reaction, and the reacti...

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Abstract

The present invention provides methods for designing and constructing novel molecular cloning vectors for cloning DNA fragments. The invention utilizes the mechanism that the expression product of the bacteriolytic gene E of the bacteriophage ΦX174 can lead to the death of the host Escherichia coli cell to design and construct the cloning vector. When the target fragment is inserted into the cloning site on the vector, it will cause the expression of the E gene to be silent, so the positive transformant containing the recombinant vector grows normally into a colony clone when it is cultivated at the induction temperature (42 ° C), and the vast majority (99.9 %) negative transformants will die due to E gene expression when cultured at this temperature, and cannot grow into colony clones. The invention can be used for designing and constructing common cloning vectors and T vectors. The cloning vector of the present invention has the technical feature of inhibiting the generation of negative clones, so it is especially suitable for constructing genome libraries and cDNA libraries in addition to being used for single gene cloning.

Description

technical field [0001] The content of the present invention belongs to the field of biotechnology. Background technique [0002] 1. Molecular cloning carrier technology: Molecular cloning technology is the basis of genetic down-programming technology. Molecular cloning technology is used to insert a target DNA fragment (or gene) into a plasmid vector (a closed circular double-stranded DNA molecule that can be replicated and passed down in bacterial cells) to preserve the target DNA segment for a long time for subsequent research. Therefore, an important technical basis in molecular cloning technology is the plasmid vector (or cloning vector) used for cloning the target DNA fragment. A cloning vector molecule must contain at least the following three functional structures: genetic elements necessary for plasmid replication, resistance genes for screening positive clones, and restriction sites for inserting target DNA fragments. [0003] 2. Molecular cloning screening techno...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 马跃
Owner SOUTHWEST UNIVERSITY
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