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PCR - Thermal Cycler Systems, Enzymes and Master Mixes

Thermo Fisher Scientific

Thermo Scientific™ Phusion™ PCR - Site-Directed Mutagenesis Kit

The Thermo Scientific™ Phusion™ Site-directed Mutagenesis Kit is a versatile and effective 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 bring 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 then be transformed into any competent E. coli cell.

The optimal annealing temperature for Phusion™ DNA Polymerases can differ significantly from that of Taq-based polymerases.

Technical Specifications
Application Note

PCR - Phusion™ Site-Directed Mutagenesis Kit

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
• Compatible with all strains of competent E. coli cells

High Fidelity PCR DNA Polymerase Enzymes

High-fidelity PCR enzymes are used for applications requiring high accuracy during DNA amplification such as cloning, sequencing or mutagenesis. Thermo Scientific Phusion high-fidelity DNA polymerases were created by fusing a dsDNA-binding domain to a Pyrococcus polymerase–like proofreading enzyme. Due to this unique protein fusion engineering, Phusion DNA Polymerases have very low error rates, fast extension rates, and high inhibitor tolerance.

	Phusion™ Plus DNA Polymerase	Phusion™ High Fidelity DNA Polymerase	Phusion™ Hot Start II DNA Polymerase	Phusion™ Flash High-Fidelity PCR Master Mix	Phusion™ U Hot Start DNA Polymerase	Phusion™ U Multiplex PCR Master Mix
*Fidelity (vs. Taq* enzyme)**	>100x	52x	52x	25x	25x	25x
T_m calcuclation required	No	Yes	Yes	Yes	Yes	Yes
Enhanced specificity (hot-start version)	Yes	No	Yes	Yes	Yes	Yes
Amplification length	Up to 20kb	Up to 20kb	Up to 20kb	Up to 20kb	Up to 20kb	Up to 2.5kb
Speed	15–30 sec/kb	15–30 sec/kb	15–30 sec/kb	<15 sec/kb	15–30 sec/kb	15–30 sec/kb
GC-rich format	Yes	Yes	Yes	No	Yes	No
dUTP incorporation	No	No	No	No	Yes	Yes
Designed for multiplex PCR	Hayır	Hayır	Hayır	Hayır	Hayır	Evet, 20 target'a kadar
Stand-alone enzyme format	Colorless	Colorless Green	Colorless Green		Colorless Green
2x master mix format	Colorless Green	Colorless with HF buffer Colorless with GC buffer	Colorless Green	Colorless	Colorless	Colorless Green

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Thermo Fisher Scientific

Thermo Scientific™ Phusion™ PCR - Site-Directed Mutagenesis Kit

PCR - Phusion™ Site-Directed Mutagenesis Kit

High Fidelity PCR DNA Polymerase Enzymes

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Thermo Fisher Scientific

Thermo Scientific™ Phusion™ PCR - Site-Directed Mutagenesis Kit

PCR - Phusion™ Site-Directed Mutagenesis Kit

High Fidelity PCR DNA Polymerase Enzymes

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