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Subcloning technology process——Generalbiosystems

Update time : 2020-08-14

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Subcloning technology process——Generalbiosystems

Subcloning technology refers to the re-cloning of gene DNA fragments in the case of obtaining them, so as to further analyze or recombine the target gene DNA. Subcloning technology is also a molecular cloning technology. 

The emergence of this technology allows people to have a more in-depth study of gene DNA sequences, clarify the function of genes, and analyze the phenotype of organisms from the genetic perspective.

The technical process of subcloning is mainly divided into four steps: obtaining the target fragment, connecting the digested vector and the target fragment, transferring into host cells, and identifying and screening.

Method of obtaining the target fragment

 Obtain the target gene fragment directly from the gene library; 

 Using PCR technology, use mRNA as a template to reverse transcribe the cDNA sequence to obtain the cDNA library to obtain the target gene; 

 Use restriction enzymes to cut the donor DNA into many fragments and introduce them into cells. After screening, cells containing the target gene are obtained; 

 Gene sequence information is obtained and artificial synthesis is performed in vitro.

Ligation of restriction digestion vector and target fragment

Choose the appropriate restriction endonuclease to cut the target fragment sequence of the vector. Under normal circumstances, there will be three situations: symmetric sticky end, asymmetric sticky end and blunt end. In subcloning experiments, asymmetric sticky end ligation is preferred, which can direct foreign DNA into the vector.

Transfer into host cells

There are usually two methods for transferring the vector linked to the target gene into cells: transformation/transfection and transduction. 

Transformation/transfection is the transfer of recombinant plasmids or bacteriophages into treated host cells; the method of transduction is to infect the host cells with viruses carrying foreign DNA. Generally, transduction is more efficient than transformation methods.

Identification screening

The screening of cloned cells usually uses direct screening. The carrier has identifiable genetic markers, which can effectively distinguish and separate recombinant molecules from original cells. 

After some carrier molecules carry foreign genes, the color of colonies or plaques formed has obvious changes, such as blue to colorless, and the obvious color phenotypic changes can clearly distinguish recombinant molecules. 

In addition, there are some other drug screening methods that can effectively screen cloned cells through phenotypic phenomena.

Generalbiosystems has an experienced technical team and a patented gene synthesis platform, which can provide customers with one-stop subcloning services including target gene synthesis, vector construction and transformation, identification and screening. 

In the subcloning service, 4μg of dry powder plasmid DNA, glycerol bacteria or puncture bacteria containing plasmids, sequencing results (ab1 format), and enzyme digestion verification documents passed QC inspection will be delivered. Generalbiosystems will provide customers with accurate and fast subcloning services.