This is the instruction manual of Agilent QuikChange II XL Site-Directed Mutagenesis Kit
The Agilent QuikChange II XL Site-Directed Mutagenesis Kit is designed for in vitro site-directed mutagenesis of large, difficult constructs and virtually any double-stranded plasmid. The kit eliminates the need for subcloning, single-stranded DNA rescue, specialized vectors, or in vitro methylation treatment steps. It utilizes a rapid three-step procedure to generate mutants with greater than 80% efficiency.
| Catalog #200521 reactions | 10 reactions |
|---|---|
| PfuUltra High Fidelity DNA polymerase | 2.5 U/ μl |
| Dpn I restriction enzyme | 10 U/ μl |
| Oligonucleotide control primer length | 34-mer |
| Mutagenic primer length range | 25 to 45 bases |
| Minimum melting temperature | ≥78°C |
| Minimum GC content | 40% |
| XL10-Gold transformation efficiency | 5-fold higher than XL1-Blue |
The pWhitescript 4.5-kb control plasmid is used to test the efficiency of mutant plasmid generation by creating a point mutation that reverts a stop codon to a glutamine codon.
The Dpn I endonuclease is specific for methylated and hemimethylated DNA with the target sequence 5´- Gm 6ATC-3´.
Primers should be 25 to 45 bases long with a melting temperature of ≥78°C, and the desired mutation should be located near the center of the primer.