Recombinational Cloning

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Recombinational cloning became popular with the introduction of three cloning systems: Gateway®, Creator™, and Echo Cloning™ systems. These systems use a site-specific recombinase (Integrase in Gateway and Cre Recombinase in Creator and Echo) to allow reliable transfer of a fragment from one vector to another without using restriction enzymes and ligases. 

Typically, a researcher would clone a sequence of interest into a holding vector ("Entry" for Gateway and "Donor" for Creator) using traditional cloning methods. Once the new clone is made, it is easily shuttled to many different "destination" or "acceptor" vectors that contain the appropriate sequence recognized by the recombinase (attachment sites attB and attP with Gateway and loxP with Creator/Echo). Higher throughput is possible with these systems and they have become a useful tool for screening many different expression hosts for protein expression projects or for multiple reporter vectors for functional analysis studies. At this time, only the Gateway system is still commercially supported, although NEB does sell Cre Recombinase (NEB #M0298), an essential reagent for the in vitro recombination step used by the Creator and Echo Cloning systems.

Advantages:
  • Allows high-throughput vector creation
  • Widely available ORF collections
Disadvantages:
  • Cost relative to traditional methods
  • Vector sets typically defined by supplier
  • Propietary enzyme mixes often required

Recombinational Cloning Workflow

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Note that times are based on estimates for moving a gene from one plasmid to another. If the source for gene transfer is gDNA, add 2 hours to calculation for the traditional cloning method. Total time does not include transformation, isolation or analysis.