The BP Clonase Enzyme Mix contains both the integrase enzyme and a co-factor protein, Integration Host Factor (IHF) that catalyzes the recombination of attB and attP sites. Figure 2: Movement of the gene of interest from the entry vector into a destination vector to generate an expression vector. This uses a similar reaction but as the initial att sites are now attL and attR sites, LR Clonase Enzyme Mix is used (Figure 2). to create an expression vector for use in your experiments). Using your entry vector you can move your gene of interest into a destination vector (e.g. The donor vector contains the toxic ccdB gene, which is replaced by your gene of interest upon successful recombination meaning all clones that result following transformation contain the entry vector with your gene of interest.įigure 1: Insertion of PCR product into donor vector to create entry clone. The recombination results in the formation of an entry vector containing your gene of interest (Figure 1). Initially, you create your PCR product with attB sites and then combine it with a donor vector containing attP sites plus BP clonase. attL and attR sites when paried together and combined with the LR Clonase Enzyme Mix will recombine into attB and attP sites.attB and attP pair together and when combined with the BP Clonase Enzyme Mix will recombine into attL and attR sites.These sites work together in pairs to allow your gene of interest to be moved between plasmids by using particular enzyme mixes: There are four different att sites: attB, attP, attL, and attR. These specific sites are known as att sites, which are targeted by the integrase enzyme. This cloning method uses site-specific recombination. īefore we provide you some top tips for successful cloning with Gateway vectors, let’s review how this technology works. I have described below an overview of the Gateway cloning system and some factors that will increase the cloning efficiency. Gateway cloning is efficient and allows you to clone without using restriction enzymes or ligation reactions, but it still has multiple steps that can be optimized. ![]() Gateway cloning gives researchers the opportunity to easily transfer DNA fragments into plasmids using proprietary recombination sites called Gateway att sites and either the LR clonase or BP clonase enzyme mixes. An efficient cloning system that is in vogue at present is the Gateway® Cloning Technology from Invitrogen.
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