Four Steps of Subcloning Technology Protocol

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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
• Subcloning Technology Protocol- connect enzyme vector and target fragment

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.

1. Subcloning Technology Protocol- transform into host cell

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.

1. Subcloning Technology Protocol-identify & screen

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.