Phusion Site-Directed Mutagenesis Kit
- Robust, reliable amplification for mutagenesis
- No special vector or site requirements
- No need to destroy the starting template
- Minimize unwanted secondary mutations
- Amplify large plasmids up to 10 kb
- Hot start prevents non-specific amplification and degradation
- No purification needed before or after ligation
Site-directed mutagenesis is widely used in the study of gene and protein functions. With the Thermo Scientific Phusion Site-Directed Mutagenesis Kit, point mutations, insertions and deletions can be introduced in any type of plasmid DNA. This kit uses the highly processive Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase for exponential PCR amplification of dsDNA plasmid to be mutated. The mutagenesis protocol comprises only three steps:
- PCR amplificaton of target plasmid with two phosphorylated primers. The primers, one or both with desired mutation(s), are designed so that they anneal back to back to the plasmid
- Circularization of mutated PCR products by ligation with T4 DNA Ligase
- Transformation to E. coli.
For the target plasmid, there are no requirements such as special vectors, restriction sites or methylation status. Because minute amounts of template DNA are exponentially amplified in this method, the fraction of non-mutated template is minimal. Thus there is no need to destroy it in a separate step.
Phusion® Hot Start II DNA Polymerase ensures high fidelity for the exponential amplification, thus reducing unwanted secondary mutations and enabling amplification of large plasmids up to 10 kb. Phusion Hot Start II DNA Polymerase combines the DNA polymerase and a reversibly bound, specific Affibody® protein1,2, which inhibits the DNA polymerase activity at ambient temperatures. The hot start modification in the polymerase prevents the amplification of nonspecific products and unwanted degradation of primers prior to the first cycle of PCR.
The Phusion Site-Directed Mutagenesis Kit includes:
- Phusion Hot Start II DNA Polymerase
- 5x Phusion HF Buffer, dNTPs
- Quick T4 DNA Ligase
- 2x Quick Ligation™ Buffer
- control plasmid with control primer mix.
The Quick T4 DNA Ligase included in the kit enables direct ligation without extra purification steps before or after the ligation. The kit is compatible with all competent E. coli cells, giving the user an option to use any cells available in the laboratory.