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  7. Thermo Scientific™ Phusion Hot Start II High-Fidelity PCR Master Mixes

Thermo Scientific™ Phusion Hot Start II High-Fidelity PCR Master Mixes

Catalog No. F566S
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F566S Green 100 Reactions
F566L Green 500 Reactions
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F565S Colorless 100 Reactions
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Catalog No. F566S Supplier Thermo Scientific™ Supplier No. F566S
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Description

Includes

Phusion Hot Start II High–Fidelity DNA Polymerase

  • Phusion Hot Start II High–Fidelity DNA Polymerase (2U/μL)
  • 5x Phusion HF Buffer
  • 5x Phusion GC Buffer
  • DMSO
  • 50mM MgCl2 solution
Phusion Green Hot Start II High–Fidelity DNA Polymerase
  • Phusion Hot Start II High–Fidelity DNA Polymerase (2U/Glc muL)
  • 5x Phusion Green HF Buffer
  • 5x Phusion Green GC Buffer
  • DMSO
  • 50mM MgCl2 solution
Both Phusion and Phusion Green Buffer provide 1.5 mM MgCl2 in the final 1x concentration

Thermo Scientific Phusion High-Fidelity DNA polymerases set a gold standard for high performance PCR.

Thermo Scientific Phusion High-Fidelity DNA Polymerases set a gold standard for high performance PCR. Featuring an error rate 50-fold lower than that of Taq, and 6-fold lower than that of Pfu, Phusion High-Fidelity DNA Polymerase is excellent choice for cloning and other applications requiring high fidelity. Phusion DNA Polymerases offer robust performance with short protocol times, even in the presence of PCR inhibitors, and generate higher yields with lower enzyme amounts than other DNA polymerase.

Highlights

  • Reaction set up at room temperature
  • No non-specific amplification and primer degradation during reaction set up
  • Zero-time reactivation due to unique hot start technology
  • High fidelity (52X Taq)
  • Fast PCR due to short extension times (15-30 s/kb)
  • Robust reactions, minimal optimization needed
  • Increased product yields with minimal enzyme amounts
  • Direct loading on gels

With Phusion Hot Start II High-Fidelity DNA Polymerases amplification proceeds without the production of nonspecific products due to the combination of Phusion DNA Polymerase and a reversibly bound, specific Affibody ligand that inhibits DNA polymerase activity at room temperature. The Affibody ligand also inhibits the 3'→5' exonuclease activity of the polymerase, thus preventing degradation of primers and template DNA during reaction set up. At temperatures that promote polymerase activity, the ligand is released, rendering the polymerase fully active. Phusion Hot Start II DNA Polymerase is immediately reactivated at high temperatures so it does not require a separate activation step in PCR protocols.

Phusion Green Hot Start II High-Fidelity PCR Master Mix is convenient 2X mix designed to minimize the number of pipetting steps. The master mix contains Phusion Hot Start II DNA Polymerase, nucleotides and optimized reaction buffer including MgCl2. The buffer also includes a density reagent and two tracking dyes for direct loading of PCR products on a gel.

Applications

  • High-fidelity PCR
  • High throughput
  • Amplification of difficult (GC-rich) templates
  • Template generation for sequencing
  • Multiplex PCR
  • Long-range PCR
  • Cloning
  • Mutagenesis
  • Microarray

Using Phusion DNA Polymerases

Annealing rules for Phusion DNA Polymerases are different from many common DNA polymerases (such as Taq DNA polymerases).

Specifications

Concentration 2X
Content And Storage • 2X Phusion Green Hot Start II High-Fidelity PCR Master Mix (provides 1.5 mM MgCl2 in the final 1X concentration)
• Nuclease-free water
• DMSO

Store at -20°C.
GC-Rich PCR Performance Medium
Polymerase Phusion Hot Start II High-Fidelity DNA Polymerase
Reaction Speed Fast
Product Type Hot Start High-Fidelity PCR Master Mix
Quantity 100 reactions
Shipping Condition Dry Ice
For Use With (Application) Hot-start PCR, High-fidelity PCR
Fidelity (vs. Taq) 52X
Hot Start Built-In Hot Start
No. of Reactions 100 Reactions
Overhang Blunt
Reaction Format SuperMix or Master Mix
Size (Final Product) 20 kb or less
Color Green
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Frequently Asked Questions (FAQs)

What is the difference between Phusion Flash High-Fidelity PCR Master Mix and Phusion Hot Start II High-Fidelity PCR Master Mix?

Phusion Hot Start II High-Fidelity PCR Master Mix is a 2X ready-to-use solution based on Phusion Hot Start II DNA Polymerase. It is designed for the highest fidelity (52X Taq) and specificity. Phusion Flash High-Fidelity PCR Master Mix is based on a modified Phusion Hot Start II DNA Polymerase, which allows for extremely short cycling times and features somewhat lower fidelity (25X Taq).
Which buffer is used in Phusion Hot Start II High-Fidelity PCR Master Mix?

Phusion Hot Start II High-Fidelity PCR Master Mix is based on Phusion HF buffer. For amplification of GC-rich targets, we recommend to add 3% DMSO.
What is enzyme concentration in Phusion Hot Start II High-Fidelity PCR Master Mix?

Phusion Hot Start II DNA polymerase concentration is optimized to give good results in most reactions. When the PCR reaction is set up according to the instructions, the final concentration of Phusion enzyme is 1 U in 50 µL reaction (0.4 U in 20 µL reaction).
Do Phusion DNA Polymerases add the non-template dependent 3'-A overhang?

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.
Can Phusion DNA Polymerases extend at 1 second/kb?

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.
Can protocols optimized for Phusion DNA Polymerase be directly applied to Phusion Hot Start II DNA Polymerase?

Yes, protocols optimized for Phusion DNA Polymerase can be applied to Phusion Hot Start II DNA Polymerase reactions.
Do Phire and Phusion Hot Start II DNA Polymerases need a separate activation step in the PCR protocol?

No separate activation step is required since Phire and Phusion Hot Start II DNA Polymerases are inactivated by a reversibly bound, specific Affibody ligand that dissociates during initial denaturation.
Can I use Phusion Green and Phire Green PCR products for subsequent applications such as sequencing, ligation, and restriction digestion?

Yes. The dyes do not interfere with downstream applications such as DNA sequencing, ligation, and restriction digestion.
What dyes are present in Phusion Green and Phire Green buffers?

Phusion Green and Phire Green buffers contain two dyes for monitoring electrophoresis progress. The blue dye migrates with 3-5 kb DNA fragments and the yellow dye migrates faster than 10 bp DNA fragments in 1% agarose gel. The dyes have excitation peaks at 424 nm and 615 nm, respectively.

For Research Use Only. Not for use in diagnostic procedures.