NZYMutagenesis kit (MB012)

Refrigerated package

114,00160,00

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.

Features: 
– 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

Applications:
– Site-directed mutagenesis in any type of plasmid DNA

Protocol overview:
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.

NZYMutagenesis_kit_protocol

Specifications:
– 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

Components:
– 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

Product Brochure EN
Safety Information EN
Informacao de Seguranca PT
Certificate of Analysis ZN111
Certificate of Analysis ZN051
Certificate of Analysis YO113
Certificate of Analysis YO112
Certificate of Analysis YO111
Certificate of Analysis YO101
Certificate of Analysis YO091

A protein trisulfide couples dissimilatory sulfate reduction to energy conservation
Santos AA, Venceslau SS, Grein F, Leavitt WD, Dahl C, Johnston DT, Pereira IA
Science, 2015

Interaction studies between periplasmic cytochromes provide insights into extracellular electron transfer pathways of Geobacter sulfurreducens
Fernandes AP, Nunes TC, Paquete CM, Salgueiro CA
Biochem J, 2017

Exploring the molecular mechanisms of electron shuttling across the microbe/metal space
Paquete CM, Fonseca BM, Cruz DR, Pereira TM, Pacheco I, Soares CM, Louro RO
Front Microbiol, 2014

Ser119 phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor
Soares-Dos-Reis R, Pessoa A S, Matos M R, Falcão M, Mendes VM, Manadas B, Monteiro F A, Lima D, Reguenga C
Biochemical Journal, 2014

Dual role of Tlx3 as modulator of Prrxl1 transcription and phosphorylation
Regadas I, Soares-Dos-Reis R, Falcão M, Matos MR, Monteiro FA, Lima D, Reguenga C
Biochimica et Biophysica Acta, 2014

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