Hi all,

I'm working on this topic alone and would love some help and guidance if possible. I'm working from this paper - https://journals.asm.org/doi/10.1128/AEM.00313-14#B3 (High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage) as we have all of the plasmids in house but no one is left who can teach it to me.

Can someone please give me a simple step by step protocol? The paper here seems to assume lots of prior assumed knowledge which is not easy to find.

Right not I have a donor plasmid which I think is used to generate an intermediate strain but I'm missing how to do this very basic step. Any help you could provide me with would be greatly appreciated.

Best

John

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