GeneArt™ Site-Directed Mutagenesis PLUS System (Invitrogen™)

The GeneArt Site-Directed Mutagenesis PLUS System brings our mutagenesis technology to the next level with the ability to perform single- or multi-site mutations in larger plasmids with greater efficiency and in less time than the competition.

• Powerful—make substitutions, deletions, or insertions of up to 3 nucleotides in 1, 2, or 3 separate sites in DNA plasmids of up to 14 kb
• Flexible—perform multi-site mutagenesis with degenerated nucleotides and single-site mutations up to 25 nucleotides long or 12 nucleotides long with degenerated nucleotides
• Efficient—obtain your desired mutants the first time; over 90% correct mutants in a 10 Kb plasmid
• Fast—obtain your mutated plasmid DNA in typically less than 3 hours with the simple, minimal-handling protocol
• Versatile—use plasmids of many sizes, and DNA isolated from any source, with no need for specialized vectors, host strains, or restriction sites

Optimized Mutagenesis Protocol
The GeneArt Site-Directed Mutagenesis System was further optimized for efficiency and multi-site capability, resulting in the 'PLUS' kit. Like the first generation GeneArt Site-Directed Mutagenesis System, DNA methylation and amplification steps are combined into a single reaction, with no need for an in vitro DpnI digestion step. After methylation and amplification, a 15 minute in vitro recombination reaction of the amplified PCR products increases the colony output by 3 to 10 fold, resulting in a higher mutagenesis efficiency. A final transformation of the mutated DNA into DH5α E.coli cells digests any methylated parental DNA, leaving behind only the intact unmethylated mutagenesis reaction product. No purification steps or additional digestions are needed. Individual colonies can be selected the following day to verify mutations.

Simple Creation of Desired Mutants
Creating mutants with the GeneArt Site-Directed Mutagenesis PLUS System relies on the inherent properties of DNA methylase, high fidelity DNA polymerase, recombination enzymes, and the native McrBC endonuclease of E. coli. Simply incorporate your desired mutation or mutations into primers using our GeneArt Design Tool for Seamless Assembly and Mutagenesis (see 'in silico Design Support', below). Combine the vector and mutagenesis primers, and after PCR, recombination, and transformation you will have vectors with only the desired mutations with over 90% efficiency.

in silico Design Support
A key step in GeneArt site-directed mutagenesis is the correct design of primers with the appropriate homology and spacing to help ensure successful mutagenesis of your plasmid. To simplify and speed the design process, we provide a free online tool, the GeneArt Design Tool for Seamless or High-Order Assembly and Mutagenesis, to help you design your experiment in silico. The tool allows you to review your plasmid sequence, designs DNA oligos for introduction of the desired mutations, provides an updated amino acid sequence for your mutated gene, displays a graphical representation of the new construct, and enables the download of an annotated sequence in GenBank format that is compatible with Vector NTI software.