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PGADT7-In vector applicable to In-Fusion cloning as well as construction and use methods of pGADT7-In vector

A construction method and pgadt7-in technology, applied in the field of pGADT7-In carrier, can solve problems such as time-consuming and achieve the effect of improving the success rate, reducing the experimental cost and shortening the experimental period

Active Publication Date: 2018-07-27
ZHOUKOU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention overcomes the cumbersome steps of enzyme cutting and ligation in the traditional vector construction process and the limitation of the enzyme cutting site, the Gateway technology requires longer primer initiation and two rounds of reaction time-consuming, and the improved Gateway technology requires Disadvantages of two-round PCR

Method used

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  • PGADT7-In vector applicable to In-Fusion cloning as well as construction and use methods of pGADT7-In vector
  • PGADT7-In vector applicable to In-Fusion cloning as well as construction and use methods of pGADT7-In vector
  • PGADT7-In vector applicable to In-Fusion cloning as well as construction and use methods of pGADT7-In vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of embodiment 1pGADT7-In carrier

[0037] Vectors pGBKT7 and pGADT7 were from Clontech. All primers in the examples were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0038] The pGBKT7 vector and the pGADT7 vector have an identical sequence of more than 15 bp upstream of the EcoRI restriction site in the multiple cloning site, but the downstream sequence of the EcoRI restriction site is different. In this example, pGADT7 was transformed by site-directed mutagenesis PCR technology. A sequence 15 bp downstream of the EcoRI restriction site of the multi-cloning site on the pGBKT7 vector was used to replace a sequence of 29 bp downstream of the EcoRI restriction site of the multi-cloning site on the pGADT7 vector to obtain the vector pGADT7-In, see figure 1 with figure 2 ,Specific steps are as follows:

[0039] Step 1: Design forward primer Yu01 and reverse primer Yu02, the 5'-CCGGGGATCCGTCGA-3' in the forward primer Yu01 is the 15bp forward ...

Embodiment 2

[0054] Example 2 SV40 large T-antigen protein, p53 protein and lamin C (66-230) Interaction verification between proteins

[0055] In this example, the proteins SV40large T-antigen, p53 and lamin C (66-230) They are abbreviated as T, 53 and Lam respectively. It is known that protein T interacts with protein 53, but protein T and protein Lam do not interact.

[0056] 1. Strain material and culture medium

[0057] Yeast strain AH109 and all yeast media were from Clontech. Competent Escherichia coli were purchased from Beijing Quanshijin Biological Company. The yeast culture temperature was 30°C, and the Escherichia coli culture temperature was 37°C. Solid culture is carried out in an incubator, and liquid culture is carried out in a constant temperature shaking shaker.

[0058] 2. Vectors and Primers

[0059] Vectors pGBKT7 and pGADT7 and vectors pGADT7-T, pGBKT7-Lam and pGBKT7-53 were from Clontech. The pGADT7-In vector is from Example 1. All primers in the examples wer...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a pGADT7-In vector applicable to In-Fusion cloning as well as construction and use methods of the pGADT7-In vector.A multiple cloning site of pGADT7 is transformed with a site-directed mutagenesis technology, the pGADT7-In vector is obtained, after pGBKT7 vector and the pGADT7-In vector are subjected to enzyme digestion with EcoRI, formed tail ends contain the same sequence with length being 15 bp or longer, and further, vector construction is performed with the In-Fusion technology; only one pair of primersis required for construction of a bait vector or a prey vector of a given gene, and one-time PCR is performed; an obtained PCR product is subjected to an In-Fusion reaction with pGBKT7 or pGADT7, escherichia coli is transformed, LB flat plates with different resistance are coated with an obtained product, identification is performed by bacterial colony or bacterial liquid PCR, and construction ofthe bait vector or the prey vector is completed easily, rapidly and efficiently. With application of the pGADT7-In vector and the vector construction method to construction of a yeast two-hybrid vector, experimental cycle is greatly shortened, experimental cost is reduced, and success rate is increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pGADT7-In carrier suitable for In-Fusion cloning and its construction and use method. Background technique [0002] Yeast two-hybrid is the most commonly used method to study protein interaction, and it is often used for verification of interaction between known proteins and screening of proteins with known protein interaction. Taking the commonly used GAL4 yeast two-hybrid system as an example, the yeast two-hybrid technology contains two vectors, which respectively contain the activation domain (AD) of the transcription factor GAL4 and the DNA binding domain (BD) of the transcription factor GAL4. The vector that fused and expressed the target gene a and AD is called a prey vector, and the vector that fused and expressed the target gene b and BD is called a bait vector. When the target gene a and the target gene b interact, the AD of GAL4 is spatially close to the BD,...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/81
CPCC12N15/63C12N15/66C12N15/81
Inventor 李成伟张菊廖立冰于德水张怡徐克东刘坤谭光轩陈璨刘霞韩霄萌张璇位张坤
Owner ZHOUKOU NORMAL UNIV
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