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Construction method of high-throughput CRISPR gene editing tool vector

A gene editing and construction method technology, applied in the field of high-throughput CRISPR gene editing tool carrier construction, can solve the problems of cumbersome steps, time-consuming, and inability to achieve high-throughput, and achieve optimized construction steps, reduced volume, and reduced experimental costs Effect

Pending Publication Date: 2021-12-03
NINGXIA MEDICAL UNIV
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Problems solved by technology

However, the disadvantages of classic vector construction are cumbersome steps, time-consuming, and inability to achieve high throughput

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  • Construction method of high-throughput CRISPR gene editing tool vector
  • Construction method of high-throughput CRISPR gene editing tool vector
  • Construction method of high-throughput CRISPR gene editing tool vector

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[0022] In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0023] A method for constructing a high-throughput CRISPR gene editing tool vector, comprising the steps of:

[0024] S1. Primer design and synthesis;

[0025] S2. Primer annealing: place the system in a PCR instrument at 100°C for 10 minutes, then let it cool down to room temperature naturally; (Primer T 1μL (100μmol / L), Primer B 1μL (100μmol / L), ddH2O 98μL, total 100μL) ;

[0026] S3, sgRNA recombinant plas...

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Abstract

The invention discloses a construction method of a high-throughput CRISPR gene editing tool vector. The construction method comprises the following steps: S1, designing and synthesizing primers; S2, annealing the primers; S3, constructing sgRNA recombinant plasmids; S4, converting to obtain 24 parts of 2 [mu]l of a mixture; S5, screening a monoclone; and S6, conducting plasmid extraction and sequencing. The method optimizes construction steps of the recombinant plasmids and incubation steps of a transformation experiment, at the same time shortens time of plasmid construction and the transformation experiment, improve the transformation efficiency and a positive rate, reduces experiment cost, replaces a traditional coating rod method in a monoclonal screening process, and lays a foundation for finally achieving automatic and high-throughput sgRNA vector construction. The method solves a contradiction between enzyme digestion and linkage, unifies a system which cannot be unified, can be used to construction of gene editing sgRNA, and also can be used to a seamless cloning technology of vector construction.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and more specifically relates to a method for constructing a high-throughput CRISPR gene editing tool vector. Background technique [0002] CRISPR-Cas9 gene editing technology is currently the most widely used gene manipulation tool. The construction of the vector connecting sgRNA is the most basic and important experimental operation of this technology. However, the disadvantages of classic vector construction are cumbersome steps, time-consuming, and inability to achieve high throughput. Therefore, we propose a high-throughput CRISPR gene editing tool vector construction method. Contents of the invention [0003] The purpose of the present invention is to solve the shortcomings in the prior art, and propose a method for constructing a high-throughput CRISPR gene editing tool vector. [0004] In order to achieve the above object, the present invention provides the following technical s...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/66
CPCC12N15/113C12N15/63C12N15/66C12N2310/20
Inventor 杨延辉林源田静何龙杨林王大军杨丽唐静李晓雨马凯杨玉玛刘河涛
Owner NINGXIA MEDICAL UNIV
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