Category: Subcloning

Four Steps of Subcloning Technology Protocol

Subcloning refers to the technique of re-cloning a DNA fragment from one vector to another, so that we can more easily perform analysis, transformation, and recombination of the target gene(s). Subcloning is an important tool in any molecular biologist’s toolkit, helping to elucidate the function of a target gene and to easily analyze its phenotype. There are four steps in the subcloning process: obtain the target fragment, connect enzyme vector and target fragment, transform in host cell, identify and screen.

  1. Subcloning Technology Protocol- obtain the target fragment

    • Find the target fragment in a gene library
    • The cDNA sequence was reverse transcribed with mRNA as a template through PCR, so we can obtain the target fragment from the cDNA library
    • Cut the DNA into many fragments with restriction enzymes and introduce into cells; we can screen for cells containing the target gene(s)
    • Synthesize the gene sequences in vitro
  2. Subcloning Technology Protocol- connect enzyme vector and target fragment

  3. Choose an appropriate restriction enzyme to cleave the target fragment from the vector. Cleavage in this way usually generates symmetrical cohesive ends, non-symmetric cohesive ends, or blunt ends. For subcloning, non-symmetric cohesive ends are preferred.

  4. Subcloning Technology Protocol- transform into host cell

  5. The two most common methods of transforming the target fragment into cells are transformation / transfection and transduction. In the first method, a recombinant plasmid or phage is simply transformed into a treated host cell; in the second, a host cell is transducted with a virus harboring exogenous DNA. In general, transduction is more efficient than transformation.

  6. Subcloning Technology Protocol-identify & screen

  7. Vectors with recognizable genetic markers can be used to help distinguish and separate cells transformed with recombinant DNA. For example, color can be used as such a marker, as the color of a colony may change in some vectors with exogenous genes. Other effective and widely used screening methods also exist, such as screening with drugs or with selective media.

With our Syno® 2.0 gene synthesis platform and experienced technical team, Synbio Technologies provides one-stop subcloning services including target gene synthesis, vector construction and transformation, identification, and screening.

Synbio Technologies’ PCR cloning and Subcloning Technology

PCR cloning and subcloning technology, first developed in the 1970s, is now a staple in every molecular biology lab in the world. Cloning allows researchers to much more easily understand gene function at a deeper level, and greatly facilitates gene editing. PCR cloning and subcloning technology is not only revolutionary for the field of biology, but has profound implications on fields like agriculture, industry, and medicine as well.

PCR cloning technology

PCR cloning technology is similar to natural DNA replication, and contains three basic reaction steps: modification-annealing-extension. We can obtain a desired target gene sequence with appropriate primer design. PCR can then be used to amplify this gene sequence, preparing it for use in cloning.

Subcloning technology

In molecular cloning, target DNA is assembled into a vector plasmid through restriction enzymes and screening. In subcloning, a gene of interest is transferred from one vector to another. Both processes consist of several key steps, such as screening of the target fragment, cloning vector preparation, transformation/transduction of the product into cells, and screening for cells containing recombinant plasmids.

Both PCR cloning and subcloning technology can insert a target gene into a plasmid of choice in vitro through recombinant technology. The main forms of target gene transfer into a plasmid are transformation and transduction. This allows researchers to have an enormous amount of customization available to them when trying to study a gene of interest, making cloning and sub-cloning two extremely powerful tools in a molecular biologist’s arsenal.

With our proprietary Syno® 2.0 gene synthesis platform, Synbio Technologies can provide one-step services for gene synthesis, vector construction, PCR cloning, and subcloning. Customers only need to offer the sequence information, and we can help design amplification primers and clone the PCR products to the specific sites of the new plasmid. We also provide sequencing services in order to confirm that the correct product was accurately synthesized.