Description: NZYMutagenesis kit provides a simple and highly efficient method to generate point mutations and delete or insert single (or multiple) nucleotides in any type of plasmid DNA using PCR. This kit contains NZYProof DNA polymerase for PCR amplification of dsDNA plasmid to be mutated. NZYProof DNA polymerase ensures high fidelity for the exponential PCR amplification, thus reducing the unwanted secondary mutations and enabling amplification of large plasmids up to 15 kb. The system requires the provision of two synthetic oligonucleotide primers containing the desired mutation.
– Easy site-directed mutagenesis using PCR
– Efficient mutagenesis of large plasmids up to 15 kb
– Generate mutants with an efficiency ≥ 90% of the colonies screened
– Site-directed mutagenesis in any type of plasmid DNA
The mutagenesis protocol includes only three steps:
1. Mutated primer design – Primers should have between 25 and 45 bases in length, with a melting temperature (Tm) of ≥78 ºC;
2. PCR amplification – Incorporation of the oligonucleotide primers with NZYProof DNA polymerase which generates a mutated plasmid containing staggered nicks;
3. Digestion with Dpn I – Digestion of PCR product with Dpn I endonuclease for elimination of the parental methylated and hemi-methylated DNA template and selection of the mutation-containing synthetic DNA (not methylated).
NZYTech provides convenient versions of the kit which include highly efficient competent cells for recovering of the mutated plasmid.
– Storage conditions: Store competent cells at -80 °C. Other kit components may be stored at -20 °C or at -80 °C
– Shipping conditions: Shipped at dry ice
– Reaction Buffer (10x)
– dNTP mix
– NZYProof DNA polymerase (2.5 U/µL)
– Dpn I (10 U/µL)
– Control plasmid
– Control primer mix
– NZYStar Competent Cells (*)
– Competent Cells Control Plasmid (0.1 ng/µL) (*)
(*) only provided in MB01202 kit