Hey all for the last few months I've been using GoldenGate assembly now construct a large mutant library for a directed evolution experiment. I'm not having success with this and the colonies I do get generally contain the wild type template.

I'm using PCR to generate the GoldenGate fragments as I have limited options in this regard. I am using a 4 fragment assembly and have tried several type IIS restriction enzymes. Any advice you could give me on the matter would be greatly appreciated.

Thanks in advance

John

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