I´ve been trying for the past weeks to create a plasmid through Gibson Assembly. My construct is pretty big (28kb). I am fragmenting a plasmid into 5 fragments and adding one extra fragment of 2kb following an example that has already worked in the literature. I am confident that the fragments are amplified properly through PCR, having the desired length, I have treated them with DPN I, and the design of the primers should be the correct one to anneal with each other through the overhangs. I am using GeneArt Gibson Assembly from Invitrogen. Any suggestions?
What kit are you using? I am using NEB-Assembly and in my experience, the overlapping regions is the most important part. You should have between 15-50 bp overlapping between the fragments
Are you getting no colonies, only colonies that are transformed with a plasmid used for construction (background), or transformants that contain incorrectly assembled plasmids?
Alexandra Johnson I am getting no colonies, I don't know if it may be the actual assembly that is not working, because when I use a plasmid with the same approximately size as my construct I do get colonies. Any suggestions?
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