Takara's Mutan-Super Express Km Kit is intended for successful site-directed mutagenesis in just one day. This site-directed mutagenesis kit is based on the Oligonucleotide-directed Dual Amber or ODA method and takes advantage of LA (Long and Accurate) PCR.
The Mutan-Super Express Km utilizes the pKF18k/19k vector system; these vectors contain dual amber stop mutations at their kanamycin-resistance gene. After a target sequence (< 2 kb) is inserted into either of these vectors, PCR is performed using an oligonucleotide containing the desired mutation and a selection primer that reverts the amber mutations on the kanamycin-resistance genes. The sequence between the two primers is then amplified to generate "Mutagenic-Selection DNA." Both strands of this amplified DNA fragment also serve as PCR primers, generating nicked double-stranded plasmid. When this nicked DNA is transformed into E. coli MV1184 (sup0 strain), the nicks are repaired. Transformants containing the desired site-specific mutation can be obtained after kanamycin drug selection. This simple site-directed mutagenesis procedure takes just one day and achieves mutation frequencies greater than 80%.