The original QuikChange Site-Directed Mutagenesis Kits eliminate the need for subcloning into M13-based bacteriophage vectors and for ss-DNA rescue. This makes site-directed mutagenesis studies simple and reliable and allows oligo-mediated introduction of site-specific mutations into virtually any double-stranded plasmid DNA.
QuikChange Site-Directed Mutagenesis Kit
The QuikChange site-directed mutagenesis kit is used to make point mutations, switch amino acids, and delete or insert single or multiple amino acids. The QuikChange site-directed mutagenesis method is performed using PfuTurbo DNA polymerase and a temperature cycler. PfuTurbo DNA polymerase replicates both plasmid strands with high fidelityll and without displacing the mutant oligonucleotide primers. The basic procedure utilizes a supercoiled double-stranded DNA (dsDNA) vector with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation (see Figure 1). The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by PfuTurbo DNA polymerase. Incorporation of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I. The Dpn I endonuclease (target sequence: 5´-Gm6ATC-3´) is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for mutation-containing synthesized DNA. DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to Dpn I digestion. The nicked vector DNA containing the desired mutations is then transformed into XL1-Blue supercompetent cells. The small amount of starting DNA template required to perform this method, the high fidelity of the PfuTurbo DNA polymerase, and the low number of thermal cycles all contribute to the high mutation efficiency and decreased potential for generating random mutations during the reaction.
Figure 1. Overview of the QuikChange Site-directed Mutagenesis Method.
QuikChange XL Site-Directed Mutagenesis Kit
Our QuikChange II XL site-directed mutagenesis kit is derived from the QuikChange II site-directed mutagenesis method. The XL version of the kit is specialized for efficient mutagenesis of large (8kb -14 kb) or otherwise difficult-to mutagenize plasmid templates and features components specifically designed for more efficient DNA replication and bacterial transformation. The QuikSolution reagent is provided to facilitate replication of large plasmids, while XL10-Gold Ultracompetent Cells have been included to ensure the highest transformation efficiencies possible. The transformation efficiency of XL10-Gold cells is 5-fold higher than the transformation efficiency of XL1-Blue cells employed in the original QuikChange kit. Moreover, XL10-Gold cells contain the Hte phenotype, which increases the transformation efficiency of larger DNA plasmids.
QuikChange Multi Site-Directed Mutagenesis Kit
The QuikChange Multi site-directed mutagenesis system offers the same benefits of speed, simplicity and reliability afforded by the original QuikChange kit, but is based on a completely novel technology with distinct advantages. The novel technology of the QuikChange Multi system allows mutagenesis at multiple sites in a single round, using a single oligonucleotide per site. The QuikChange Multi site-directed mutagenesis system also makes it easy to randomize key amino acids using oligos containing degenerate codons.5 No specialized vectors or unique restriction sites are needed to use the QuikChange Multi kit–virtually any plasmid of up to 8 kb is a suitable template. The rapid three-step procedure introduces mutations at three different sites simultaneously in the 4-kb QuikChange Multi control plasmid with greater than 50% efficiency.
For Research Use Only. Not for use in diagnostic procedures