Thermo Scientific™ Phusion™ Site-Directed Mutagenesis Kit with DH10B Competent Cells

Thermo Scientific™ Phusion™ Site-Directed Mutagenesis Kit with DH10B Competent Cells

The Thermo Scientific Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions into 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 T4 DNA Ligase. The resulting plasmid can be then transformed into the E.coli cells. DH10B Competent Cells are recommended for use with the kit.

Features

• 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
• Phusion Hot Start II High Fidelity DNA Polymerase minimizes unwanted secondary mutations
• Amplification of large plasmids up to 10 kb
• Hot start modification of the polymerase prevents amplification of non-specific products and unwanted degradation of primers prior to first cycle of PCR
• T4 DNA Ligase included in the kit; no purification steps before or after ligation

Contents

Box 1

• 10 x DH10B Competent Cells, 100 µL
• pUC19 DNA, 50 µL, 100 pg/µL
• S.O.C. Medium, 5 mL

Box 2

• Phusion Hot Start II DNA Polymerase, 10 µL, 2 U/µL
• Phusion HF Buffer (5X), 1.5 mL
• dNTP Mix, 10 mM each, 20 µL
• FastDigest DpnI, 20 µL
• Control plasmid (in TE buffer), 5 ng/µL
• Control primer mix (25 μM each of 5' phosphorylated primers), 20 µL
• T4 DNA Ligase, 15 µL
• Rapid Ligation Buffer (5X), 200 µL