For various mutations, such as substitutions, insertions, and deletion mutations.This kit is an inverse PCR (iPCR)-based site-directed mutagenesis kit using KOD DNA polymerase##<1) 2)="">### as a high-fidelity PCR enzyme. This reagent was developed based on a high fidelity and efficient PCR reagent, KOD-Plus- (Code No. KOD-201), which consists of KOD DNA polymerase and anti-KOD DNA polymerase antibodies##<3)>### for Hot Start PCR. This kit enables not only the introduction of point mutations, but also the introduction of large insertions and deletions. The PCR fidelity of KOD-Plus- is greater than Taq DNA polymerase (ca. 80-fold); therefore, unexpected, 2nd-site mutations can be reduced. PCR reactions can be performed using standard PCR primers and do not require phosphorylated primers, because thisprotocol contains a Phosphorylation Step of PCR products. Features: High efficiency (95% maximum) can be obtained. Simple protocol facilitates speedy experiments. Phosphorylated primers are not required.Application: *Site-directed mutagenesisSubstitution (e.g. Point mutation, Point mutation library)Deletion (e.g. 1 bp ? several kbp deletion) Insertion (e.g. Introduction of His-tag sequence)Principle: A) Inverse PCR of plasmid DNA, using a mutation primer.B) Plasmid DNA is digested by Dpn I.Note: Dpn I digests methylated DNA, such as plasmid DNA from typical E. coli cell lines (e.g. JM109 and DH5α).C) Self-ligation of PCR products is performed by a reaction with T4 polynucleotide kinase and ligase.D) Transformation of E. coli cell lines using self-ligated PCR products.