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GeneArt® Site-Directed Mutagenesis System

Catalog number: A13282

Invitrogen™  Related applications: Mutagenesis

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Description

The GeneArt® Site-Directed Mutagenesis System provides a state-of-the-art, simple, convenient, and highly efficient means to generate mutations on a target construct in vitro in less than three hours. This system replaces the popular GeneTailor™ Site-Directed Mutagenesis System, and has been completely redesigned to be at the leading edge of commercial site-directed mutagenesis products currently available on the market. This product brings our mutagenesis technology to the next level. Note: A PCR enzyme is not included with the system and must be purchased separately. The recommended enzyme for this kit is AccuPrime™ Pfx DNA Polymerase.

Powerful – Make substitutions, deletions, or insertions of up to 12 nucleotides in plasmids up to 14 Kb
Efficient – Enables you to obtain your desired mutant the first time; over 90% correct mutants for a 3 Kb plasmid
Fast – Have your mutated plasmid DNA in less then 3 hours with the simple, minimal handling protocol
Versatile – Use plasmids of many sizes, and DNA isolated from any source, with no need for specialized vectors, host strains, or restriction sites

Simple Creation of Desired Mutants
Creating mutants with the GeneArt® Site-Directed Mutagenesis System relies on the inherent properties of DNA methylase, high fidelity DNA polymerase, recombination enzymes, and the native McrBC endonuclease of E. coli. Simply incorporate the mutation or mutations (up to 12 nucleotides), that you want into primers, and after PCR, recombination, and transformation you get vectors with only the mutations you desired with up to 90% efficiency. The template vector that you add mutations to can be isolated from any source and up to 14Kb in size. For creating complete constructs, or for joining large pieces of unrelated DNA see our GeneArt® Seamless Cloning and Assembly Kit (cat# A13288) or our GeneArt® High-Order Genetic Assembly System (cat#A13286).

Optimized Mutagenesis Protocol
The GeneArt® Site-Directed Mutagenesis System has been optimized for efficiency and simplicity. The DNA methylation and amplification steps are combined into a single reaction with no need for an in vitro DpnI digestion step. After methylation and amplification, a 10 minute in vitro recombination reaction of the amplified PCR products increases the colony output by 3 to 10 fold; resulting in a higher mutagenesis efficiency. A final transformation of the mutated DNA into DH5α™ E.coli cells digests any methylated parental DNA, leaving behind only the intact unmethylated mutagenesis reaction product. No purification steps or additional digestions are needed. Individual colonies can be selected the following day to verify mutations.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Mutagenesis Capability: Single-Site Mutagenesis
Bacterial or Yeast Strain: DH5α™
Mutagenesis Efficiency: 90%
Mutagenized Region Length (up to): 12 nucleotides
Mutation Type: Deletions, Insertions, Substitutions
Plasmid Type: Plasmids up to 14 kb
Product Size: 16 reactions

Contents & storage

Each GeneArt® Site-Directed Mutagenesis System includes enough reagents for 16 reactions and a control: DNA methylase, S-adenosylmethionine, enzyme mix and buffer, enhancer, 0.5M EDTA, sterile water, pUC19WHITE mutagenesis control, and One Shot® MAX Efficiency DH5α™-T1R chemically competent cells.

A PCR enzyme is not included with the system and must be purchased separately. The recommended enzyme for this kit is AccuPrime™ Pfx DNA Polymerase.

Store the -80°C Module and the E.coli competent cells at -80°C.

Store all other components at -20°C.

Guaranteed stable for 6 months when properly stored.

Documents

Manuals & protocols