Molecular cloning allows for the assembling of recombinant DNA in a vector, typically a plasmid vector, to express target genes in a host organism. Molecular cloning is the first step in virtually any molecular biology experiment, including PCR, mutant analysis, protein tagging and protein purification.
The different cloning systems
Cloning kits provide ligase, plasmid vectors and all the reagent you need to start your cloning experiments. Cloning products are generally divided into the different categories based on the cloning methods used:
Classic ligation-based cloning
TOPO® systems (proprietary)
Ligation cloning is an efficient and cost-effective solution for standard cloning applications. It is based on the use of vectors containing multiple cloning sites: sections of the vector plasmid containing restriction enzyme cutting sites. This means that the choice of vector, for both expression and subcloning, will not only depend on your application but will also influence your PCR strategy, as you will need to insert compatible restriction enzyme endings on your amplified fragment.
Moreover, restriction digestions can create sticky (cohesive) and blunt ends: depending on the cloning strategy. Conveniently, there are dedicated kits for both blunt and sticky end ligations.
Whether you plan on cloning the same fragment into different expression vectors, fusing the same protein on different tags, or doing an extensive promoter swap experiment, it is a good idea to use a recombination-based system like Gateway® cloning. Gateway® is based on recombination rather than restriction and ligation and allows for the “swapping” of inserts between subcloning vectors (called pDONR vectors) and expression vectors. This method also allows for the combination of multiple donor vectors to create complex fusion proteins.
TOPO® cloning is based on a different concept but allows reusing vectors for multiple clones as in the Gateway system.
Any issues which arise during your experiment can be tackled with our Molecular Cloning Troubleshoot.
Video and Image Credits
Video: The University of Sydney/YouTube