I am attempting to clone a mutant gene into a large (~22 kb) plasmid containing a viral genome and just recently realized that the established method of restriction cloning that was used successfully in the past will not work in this instance due to the location of one of the cut sites. My immediate thought is to use Gibson assembly and forego restriction enzymes altogether, though I am hesitant because I don't have any experience using Gibson in such large constructs. Does anyone have any experience with this or similar situations (large expression vectors, BACs, etc.)? Any contributions are helpful
As far as I see from the description of the Gibson assembly (https://www.addgene.org/protocols/gibson-assembly/), then you will have to:
1) PCR amplify your whole 22 kb vector which may be challenging
2) in principle, you then should also sequence the whole vector as well to verify that no changes were introduced anywhere by PCR.
Since you plan to use PCR anyway (rather than only cut/paste by restriction/ligation), you may want to just remove the unwanted restriction site(s) from the insert by the overlap extension PCR(s) and then just subclone your insert in as usual.
Michael Koksharov thanks for your thoughts! I’m not worried about PCR amplifying the vector. In the past I have just amplified vectors in culture, then linearized by restriction digest. What I am most concerned about are potential issues with insertion efficiency and/or unwanted off target insertions, exonuclease activity, etc
I used Gibson assembly once for my plasmid but I was trying to generate a library of compounds and I did not get enough efficiency, but I did get some clones. You can also try some overlapping PCR steps to generate some of the inserts and then, RD/ligation.
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