GeneMorph II EZClone Domain Mutagenesis Kit INSTRUCTION MANUAL provides the usage manual of EZClone Domain Mutagenesis Kit for laboratory
The GeneMorph II EZClone Domain Mutagenesis Kit offers an efficient method for performing targeted random mutagenesis on specific protein domains or promoter elements. It utilizes a unique cloning method that eliminates the need for restriction sites or sub-cloning, allowing researchers to target functional regions easily.
The kit includes Mutazyme II DNA polymerase, which introduces mutations during PCR with a uniform spectrum, and XL10-Gold ultracompetent cells to maximize library diversity. The process involves synthesizing mutant megaprimers via error-prone PCR, followed by an EZClone reaction that anneals these primers to the donor plasmid and extends them using a high-fidelity enzyme mix before transformation into E. coli.
| Mutazyme II DNA polymerase concentration | 2.5 U/ul |
|---|---|
| 10x Mutazyme II reaction buffer volume | 150 ul |
| 2x EZClone enzyme mix volume | 250 ul |
| Dpn I restriction enzyme quantity | 100 U |
| EZClone solution volume | 30 ul |
| Positive control plasmid quantity | 210 ng |
| XL10-Gold ultracompetent cells volume | 4x 135 ul |
Sufficient reagents are provided for 10 reactions, which includes 3 control reactions.
XL10-Gold ultracompetent cells must be stored immediately at -80°C and should not be placed in liquid nitrogen.
Once thawed, full activity is guaranteed for 3 months.