I have a plasmid that I want to delete a segment from it. I designed primers for linearization of the vector without the unwanted sequence, and with overlapped ends.
I heard that Gibsson assembly should not work on a single fragment and I could not recieve an answer from the production company.
You really don't need to do gibson assembly for this at all. You should just design your primers to PCR amplify out the sequence you want and do an in vivo ligation into your bacteria. See this paper: Article PCR-mediated deletion of plasmid DNA
Wow, never heard about it. actually, I tried to do that with Gibson and it worked but with much lower efficiency than a normal Gibson assembly with 2 fragments.
Thank you so much!
They do not recommend this because it is possible to generate "mega" plasmids where the plasmid ligates to two or more copies of the plasmid
Thanks for replying, but I don't think this is the reason.
First, mega plasmids are possible also when you use a vector and an insert (5'-vector-insert-vector-insert'-3', etc.).
Second, these plasmids will not survive in the bacteria after transfection. bacteria won't amplify plasmid with the same origin of replication more than a single time.
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