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TOOLSite-Directed Mutagenesis Kit is designed for rapid site-directed mutagenesis based on new rapid cloning technology. The amplified target fragments are digested by DpnI, cyclized by Toolsitase/buffer, and then transformed to E. coli to finish the site-directed mutagenesis. The kit consists of two modules: TOOLS Ultra High Fidelity DNA Polymerase amplification module and rapid cloning module. The ultra-fidelity of TOOLS Ultra High Fidelity DNA Polymerase can significantly reduce the possibility of unexpected mutations during amplification of up to 20 kb in length. The rapid cloning module allows homologous recombination reactions to replace the conventional annealing ring-forming reactions, requiring much less template and more flexible primer design strategy. Due to the efficient seamless splicing of the two PCR products by Toolsitase technology, the kit can complete the two separate sites mutation by single amplification reaction. DpnI digested products of the specific amplicons can be directly added into recombination reaction without purification. Highly optimized reaction buffer, fast operating procedures and amazing site-directed mutagenesis efficiency make TOOLSite-Directed Mutagenesis Kit the preferred kit for DNA site mutation.