Thermo Fisher Scientific GeneArt Site-Directed Mutagenesis PLUS System

Invitrogen™ GeneArt™ Site-Directed Mutagenesis PLUS System

The GeneArt Site-Directed Mutagenesis PLUS System brings mutagenesis technology to the next level, enabling single- or multi-site mutations in larger plasmids with greater efficiency and in less time than competing methods.

주요 특징

• Powerful — substitutions, deletions, or insertions up to 3 nucleotides at 1–3 separate sites in plasmids up to 14 kb
• Flexible — multi-site mutagenesis with degenerated nucleotides; single-site mutations up to 25 nucleotides long (or 12 with degenerated bases)
• Efficient — >90% correct mutants in a 10 kb plasmid
• Fast — mutated plasmid DNA typically obtained in <3 hours with minimal handling
• Versatile — compatible with various plasmid sizes and DNA sources; no specialized vectors or host strains required

Optimized Mutagenesis Protocol

The GeneArt Site-Directed Mutagenesis PLUS System integrates DNA methylation and amplification into a single reaction, eliminating the need for in vitro DpnI digestion. After amplification, a 15-minute recombination step increases colony output 3–10×, enhancing mutagenesis efficiency. Transformation into DH5α E. coli removes methylated parental DNA, leaving only the desired unmethylated mutant plasmid. No purification or additional digestion required; colonies can be screened the next day.

Simple Creation of Desired Mutants

Mutant creation utilizes DNA methylase, high-fidelity DNA polymerase, recombination enzymes, and the native E. coli McrBC endonuclease. Desired mutations are incorporated into primers using the GeneArt Design Tool for Seamless Assembly and Mutagenesis. After PCR, recombination, and transformation, vectors containing only the desired mutations are obtained with >90% efficiency.

in silico Design Support

Primer design is crucial for successful mutagenesis. The free GeneArt Design Tool for Seamless or High-Order Assembly and Mutagenesis assists in designing primers with optimal homology and spacing. It enables plasmid sequence review, oligo design, amino acid sequence updates, graphical construct visualization, and annotated sequence downloads compatible with Vector NTI software.

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