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. Creation of Expression Clones - LR ReactionĮntry Vectors and Donor Vectors are used to capture a gene or gene fragment of interest, to create an Entry Clone.Designing a Gateway Cloning Experiment Gateway cloning proceeds in two steps: These two recombination events are perpetually reversible. Since Gateway Cloning is moving elements from one plasmid to another, they do not refer to integration and excision, but rather the BP reaction (BP→ LR) and the LR reaction (LR→ BP). The Gateway system uses both the integration and excision reactions to create an alternative to restriction enzyme cloning. The phage then begins its lytic growth cycle. Upon excision, attL and attR are converted back to attP and attB, and the phage DNA is removed from the bacterial chromosome. If the phage senses that the bacteria is under stress, it will excise itself.
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.
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