Gateway cloning takes advantage of both positive and negative selection during the process of propagating vectors and selecting for recombinant plasmids.įor instance, the Donor Vector pDONR201 contains multiple genes for selection including: your fragment of interest is now located between attL sites, and ready for subsequent Gateway Reactions. The product you generate with your Entry Vector or Donor vector is an Entry Clone i.e.
In contrast, Donor vectors require you to use PCR to add attB sites to your fragment of interest then use the Gateway BP Clonase reaction. An entry clone is a plasmid carrying a fragment of interest located between attL sites.Įntry vectors depend on conventional restriction enzyme cloning to introduce your fragment of interest between the attL sites.
After insertion, the recombination sequences are now called attL (left) and attR (right). Recombination between the B and P elements results in new recombination sequences flanking the inserted phage DNA. In this diagram, you can see that the integration system involves the specific DNA sequences found on the phage, attP-sites, as well as sites found on the bacterial genomic target, attB. Diagram of phage lambda behavior as part of a gateway cloning process.