I currently am attempting to insert a new promoter sequence ahead of my gene of interest using the Gateway cloning method (Tol2Kit: Article The Tol2kit: A multisite gateway-based construction Kit for ...
). The final stage of this method (the LR reaction) uses several entry vectors, each of which contain either the promoter, gene, or 3' terminal sequences, which are combine specifically into a destination vector to form the final construct. I currently have my final pDestination vector which has my gene of interest (gene X) under a specific promoter (promoter A). I want to replace the exiting promoter with a new one (promoter B). Can I repeat the LR reaction with only the entry vector for p5E-Promoter B and pDest-PromoterA:geneX? I don't have access to the original entry vectors currently, otherwise I would just set the entire reaction up again. I am unsure if the attB4/attB1 sites will be able to recombine again. Thank you all for your insight.
I'm not entirely sure but I think it won't work. If I understand correctly, for LR reaction to work you need to have your entry vector with attL-attL, and your destination vector with attR-ccDb-attR. In your case you lost ccdb, because you already have geneX in place of that, no?
That is correct. So i currently have an expression clone that is attb4-> promoter-> attb1-> rest of the construct. As the Attb sites are really just attR+attL sites, thats why I am even entertaining the notion if re-using the existing expression clone as a destination vector. I suppose another way of asking the question is “Are the AttB sites still able to recombine following their initial formation”. I sincerly appreciate your consideration.
Hmmmm... in that case it just might work... The recombinases (or whatever they have in the mix) might be able to bind and do their work. if you dare and try it, please let me know if it worked! I'd appreciate it:)
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