This manual provides protocols and guidelines for using the QuikChange Site-Directed Mutagenesis Kit (Catalog #200518) to perform site-specific mutations in…
The QuikChange Site-Directed Mutagenesis Kit allows for site-specific mutation in virtually any double-stranded plasmid, eliminating the need for subcloning into M13-based vectors or single-stranded DNA rescue. The system utilizes PfuTurbo DNA polymerase and a temperature cycler to generate mutants with greater than 80% efficiency using either miniprep or cesium-chloride-purified DNA.
| PfuTurbo DNA polymerase activity (30 reactions) | 80 U |
|---|---|
| Dpn I restriction enzyme activity (30 reactions) | 300 U |
| Oligonucleotide control primer length | 34-mer |
| Minimum primer length for design | 25 bases |
| Maximum primer length for design | 45 bases |
| Minimum melting temperature for primers | 78°C |
| Minimum GC content for primers | 40% |
The protocol generates mutants with greater than 80% efficiency.
The method uses either miniprep plasmid DNA or cesium-chloride-purified DNA.
The product is treated with Dpn I endonuclease to digest the parental DNA template.
The nicked vector DNA is transformed into XL1-Blue supercompetent cells.