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High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors


QuikChange XL: a highly versatile kit for site-directed mutagenesis of large plasmids

Michael Borns Peter Vaillancourt Holly Hogrefe Jeff Braman
Stratagene

The QuikChange site-directed mutagenesis kit* characteristically generates mutagenesis efficiencies exceeding 80%. We have created a new kit for mutagenesis of large plasmid templates. The QuikChange XL site-directed mutagenesis kit has been specifically formulated for mutagenesis of longer targets, and is packaged with XL10-Gold ultra competent cells**, which are optimized for the transformation of larger DNA molecules. We demonstrate the improved performance of the QuikChangeXL kit by mutagenizing a 10.1-kb retroviral vector that was otherwise difficult to mutagenize.

Site-directed mutagenesis is routinely employed in numerous research endeavors. The procedure can be laborious and time-consuming, requiring either single-stranded DNA templates1, subcloning into specialized vectors2, plasmid vectors containing unique restriction sites, and/or multiple transformations into different host strains3. In contrast, the QuikChange site-directed mutagenesis kit4 features a highly efficient procedure that is significantly streamlined when compared to other mutagenesis methods. With this kit, site-specific mutation can take place in virtually any double-stranded plasmid, eliminating the need for subcloning into M13-based bacteriophage vectors and ssDNA rescue. In addition, the kits procedure does not require specialized vectors, unique restriction sites, or multiple transformations. The protocol can be completed in just one day and generates mutagenesis efficiencies exceeding 80%.

Another important feature of the kit is the use of high-fidelity PfuTurbo DNA polymerase.*** This key component permits template DNA replicatio
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