DNA Subcloning

Moving a cloned DNA fragment from one plasmid to another is called subcloning. Subcloning is often necessary when the original cloning vector does not support applications for further study, such as expression analysis or DNA sequencing. Clearly, choosing the optimal portion of the cloned fragment of interest is extremely important in subcloning, but you also need to carefully choose the best vector for your downstream application and the technique used for subcloning. There are a number of different techniques that can be used to subclone DNA, all of which are supported by Life Technologies products.

Restriction enzyme digestion and ligation

This classic method consists of simply digesting both plasmid and fragment DNA with restriction enzymes that leave compatible nucleotide overhangs on both DNA molecules that are easily ligated to form a circular plasmid containing the fragment of interest.

TA Cloning

PCR products amplified using Taq DNA polymerase typically have 3′-A overhangs due to the terminal transferase activity of the enzyme.

Taq-amplified PCR products with 3´-A overhangs allows for efficient ligation and cloning. Invitrogen's pCR vector backbones offer kanamycin or ampicillin selection and blue/white colony screening.

Recombination Cloning

Gateway® recombination cloning technology circumvents traditional restriction enzyme based cloning limitations, enabling you to access virtually any expression system.

Seamless Cloning

Optimized to clone up to 4 DNA fragments simultaneously into virtually any linearized E.coli vector in a 30 minute room temperature reaction (up to 13 kb total size).

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