概要
This skill provides a structured framework for designing primers used in inserting DNA sequences into existing plasmids, specifically optimized for methods like NEB's Q5 Site-Directed Mutagenesis kit. It guides users through a rigorous procedural workflow—from identifying insertion boundaries to simulating PCR products—ensuring that all designs meet critical criteria like minimum 3' annealing length and precise Tm requirements. By implementing multi-step verification strategies and highlighting common molecular biology pitfalls, the skill helps researchers and bioinformaticians avoid design errors that lead to failed laboratory experiments.
