Phusion^® Site-Directed Mutagenesis Kit

A fast and reliable system for generating mutations

Note: Starting May 2010 the Phusion Hot Start DNA polymerase in the kit will be replaced with the new Phusion Hot Start II DNA Polymerase. The new polymerase allows more flexibility to primer design. No changes are needed to excisting protocols or primers.

Description

The Phusion® Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions or deletions in any type of plasmid DNA. With this kit, the entire plasmid is amplified using phosphorylated primers that introduce the desired changes. The amplified, linear PCR product, containing the desired mutation, is circularized in a 5-minute ligation reaction with Quick T4 DNA Ligase. The resulting plasmid can be then transformed into any competent E. coli cells.

General information about Phusion DNA Polymerases, their advantages and applications can be found on Phusion DNA Polymerase products page.

The Phusion^® Site-Directed Mutagenesis Protocol

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Advantages

• Robust and reliable exponential amplification method
• No requirements such as special vectors, restriction sites or methylation status for the target plasmid
• No need to destroy the starting template in a separate step
• Due to the high fidelity of Phusion® Hot Start II DNA Polymerase unwanted secondary mutations are minimized
• Amplification of large plasmids up to 10 kb
• The hot start modification in the polymerase prevents the amplification of non-specific products and unwanted degradation of primers prior to first cycle of PCR
• Quick T4 DNA Ligase included in the kit; no purification steps before or after the ligation
• Compatible with all strains of competent E. coli cells

Contents

The Phusion® Site-Directed Mutagenesis Kit includes Phusion Hot Start II DNA Polymerase, 5x Phusion HF Buffer, dNTP mix, Quick T4 DNA Ligase (NEB), 2x Quick Ligation Buffer (NEB) and a Control Plasmid with Control Primer Mix.

Storage and stability

Recommended storage temperature -20 °C. Stable for one year from the packaging date.

Ordering information

Phusion® Site-Directed Mutagenesis Kit
F-541	20 reactions including 10 control reactions