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Phusion Site-Directed Mutagenesis Kit
Thermo Scientific Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions inRead more
| Catalog Number | Quantity |
|---|---|
| F541 | 20 reactions |
Catalog number F541
Price (EUR)
255,00
Each
-
Quantity:
20 reactions
Price (EUR)
255,00
Each
Thermo Scientific 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 T4 DNA Ligase. The resulting plasmid can be then transformed into any competent E. coli cells.
The optimal annealing temperature for Phusion DNA Polymerases may differ significantly from that of Taq-based polymerases.
For optimal results start by accurately calculating your Tm with our Tm calculator.
Highlights
• 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
The optimal annealing temperature for Phusion DNA Polymerases may differ significantly from that of Taq-based polymerases.
For optimal results start by accurately calculating your Tm with our Tm calculator.
Highlights
• 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
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Fidelity (vs. Taq)52X
FormatKit
Hot StartYes
Mutagenesis CapabilitySingle-Site Mutagenesis
No. of Reactions20 Reactions
PolymerasePhusion Hot Start II High Fidelity DNA Polymerase
Product LinePhusion
Product TypeSite-Directed Mutagenesis Kit
Quantity20 reactions
Starting MaterialDNA
FormSolution
GC-Rich PCR PerformanceHigh
Reaction SpeedFast
Unit SizeEach
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Figures
The Phusion Site-Directed Mutagenesis Protocol
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Lot #Certificate TypeDateCatalog Number(s)
3260867Certificate of AnalysisJun 13, 2025F541
3255006Certificate of AnalysisJun 05, 2025F541
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Frequently asked questions (FAQs)
Phusion DNA Polymerases generate blunt end products; therefore, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product with e.g. Taq DNA Polymerase (Cat. No. EP0401). However, before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again.
Yes it is possible, especially when amplifying smaller amplicons. Processivity of Phusion DNA Polymerases is 10 times that of Pfu. We recommend extension times of 15 s/kb for Phusion Flash PCR Master Mix. 15 s/kb is a conservative value that we can promise to work with almost any amplicon. In many cases, significantly shorter extension times (0-5 s/kb) can be used without compromising the yield. What separates Phusion Flash DNA Polymerase from other fast polymerases is that all steps in the PCR protocol can be shortened, including annealing and denaturation. This results in extremely fast protocols as compared with any other polymerase.
In reference to the Phusion Site-Directed Mutagenesis Kit User Guide, we consider a short/point mutation to be 2-3 nucleotides.