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Construction method of recombinant plasmid vector p18-kan+/- containing loxP-kanr-loxP segments

A loxp-kanr-loxp, p18-kan technology, applied in the field of recombinant plasmid vector construction, can solve problems such as experiment failure, connection failure, PCR mismatch, etc.

Inactive Publication Date: 2011-11-23
INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual operation, due to insufficient PCR mismatch and primer specificity, the connection fails or the product gene is inactivated, which eventually leads to the failure of the experiment.

Method used

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  • Construction method of recombinant plasmid vector p18-kan+/- containing loxP-kanr-loxP segments
  • Construction method of recombinant plasmid vector p18-kan+/- containing loxP-kanr-loxP segments
  • Construction method of recombinant plasmid vector p18-kan+/- containing loxP-kanr-loxP segments

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Experimental program
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specific Embodiment approach 1

[0008] Specific embodiment one: this embodiment contains loxP-kan r The recombinant plasmid vector p18-kan of -loxP fragment ± The construction method is carried out according to the following steps: One, adopt PCR technique to obtain loxP-kan from pUG6 plasmid r -loxP fragment; 2. Gel recovery of 1.6Kb loxP-kan r -loxP fragment, then use the 2.7Kb pMD18-T vector as the backbone, and connect the T-A end to obtain the ligation product; 3. The ligation product is transformed into E. coli DH5α competent cells, and then spread on ampicillin-resistant LB solid medium Carry out culture, pick positive clones, inoculate in ampicillin-resistant LB liquid medium and shake culture, use alkaline lysis method to extract recombinant plasmid, that is, complete the loxP-kan containing r The recombinant plasmid vector p18-kan of -loxP fragment ± build.

[0009] In this embodiment, the pUG6 plasmid, Escherichia coli DH5α competent cells, and pMD18-T vectors are all purchased (NewEngland Bio...

specific Embodiment approach 2

[0050] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the reaction system of PCR in step one is a 18 μ L reaction system, which consists of the following components:

[0051]

[0052]

[0053] The PCR conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 10 s, annealing at 57°C for 15 s, extension at 72°C for 2 min, a total of 30 cycles, extension at 72°C for 10 min, and incubation at 4°C. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0054] Specific embodiment three: the difference between this embodiment and specific embodiment one is that the system connected in step two is as follows:

[0055]

[0056] The connection conditions are: 16°C for 3h. Other steps and parameters are the same as those in Embodiment 1.

[0057] Solution 1 in this embodiment is a commercial product, purchased from TAKARA Company, which is a mixture of ligase and buffer, purchased as a standard reagent in the kit, and used according to the instructions.

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Abstract

The invention provides a construction method of a recombinant plasmid vector p18-kan+ / - containing loxP-kanr-loxP segments, which relates to a construction method of a recombinant plasmid vector. The method comprises the following steps: 1, acquiring loxP-kanr-loxP segments from a pUG6 plasmid; 2, acquiring a connection product through T-A terminal connection by using a pMD18-T vector as the framework; and 3, converting the connection product into competent cells, picking positive ones for cloning, and extracting the recombinant plasmid. By using the pMD18-T vector as the template, loxP-kanr-loxP gene segments are introduced onto multiple cloning sites of the template, and a pair of recombinant plasmids having opposite connection directions is simultaneously constructed. Thus, both ends of the loxP-kanr-loxP site of each plasmid are provided with multiple common enzyme cutting sites, so that an exogenous gene can be conveniently connected with the loxP-kanr-loxP segments, thereby providing a new operating way for the gene knockout and replacement of eucaryotes.

Description

technical field [0001] The invention relates to a method for constructing a recombinant plasmid vector. Background technique [0002] In the process of eukaryotic gene knockout, replacement and other experiments, kan is often used r The gene is used as a screening marker, and the loxP site is used as a tool site for removing the resistance marker gene. In practical applications, this functional fragment is usually recombined with the target gene by means of complementary PCR such as fusion PCR. However, in actual operation, due to insufficient PCR mismatch and primer specificity, the ligation fails or the product gene is inactivated, which eventually leads to the failure of the experiment. Contents of the invention [0003] The object of the present invention is to provide a kind of containing loxP-kan r The recombinant plasmid vector p18-kan of -loxP fragment ± The construction method. [0004] Contains loxP-kan r The recombinant plasmid vector p18-kan of -loxP frag...

Claims

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Application Information

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IPC IPC(8): C12N15/64
Inventor 原韬沙长青谷军张晓彦陈兵赵晓龙
Owner INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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