Method for quickly constructing recombinant plasmid

Abstract

The invention belongs to the technical field of genetic engineering and discloses a method for quickly constructing a recombinant plasmid; the method comprises the steps of designing homologous recombinant primers, preparing E. coli competent cells carrying pKD46 plasmid, and co-converting an exogenous DNA fragment having sections homologous to the plasmid. An E. coli strain with Red recombinant system is constructed by using pKD46 plasmid to stably express Red recombinase under the induction of L-arabinose, the recombinase is capable of promoting the sections homologous to the plasmid and carried to two ends of the exogenous DNA fragment to recombine with a linear plasmid, it is achieved for the first time that an exogenous DNA fragment is inserted into a single limited restriction enzymes site of a support by means of RED recombination technology, it is possible to eliminate pKD46 plasmid by cultivation at 37 DEG C, no disturbance is caused to subsequent experiments, the construction steps of the recombinant plasmid are greatly simplified, and construction time is shortened. The produced recombinant plasmid is verified via PCR (polymerase chain reaction) and enzyme digestion to exhibit high positive cloning efficiency, and the method may act as a new means to construct a recombinant plasmid.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for rapidly constructing recombinant plasmids of Escherichia coli. Background technique [0002] One of the most basic techniques in genetic engineering is to construct a recombinant plasmid, which requires the integration of the target gene into the vector plasmid. The construction of conventional recombinant plasmids requires about five steps: 1. Obtaining the target DNA fragment, 2. Double digestion of the vector and the target DNA fragment, 3. In vitro ligation of the vector and the target DNA double digestion fragment, 4. Transformation of the ligation product 5. The screening of recombinants generally takes 3-5 days. Reliance on enzyme cutting sites, cumbersome experimental operations, and low recombination efficiency greatly limit the development of functional genomics research. [0003] With the development of molecular biology, a homolog...

AI Technical Summary

Problems solved by technology

The construction of conventional recombinant plasmids requires about five steps: 1. Obtaining the target DNA fragment, 2. Double digestion of the vector and the target DNA fragment, 3. In vitro ligation of the vector and the target DNA double digestion fragment, 4. Transformation of the ligation product 5. The screening of recombinants generally takes 3-5 days, the dependence of enzyme cutting sites, tedious experimental operations, and low recombination efficiency greatly limit the development of functional genomics research

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