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  7. Thermo Scientific™ Phusion U Hot Start DNA Polymerases

Thermo Scientific™ Phusion U Hot Start DNA Polymerases

Catalog No. F555S
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F555S Colorless 100 Reactions
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Description

Thermo Scientific Phusion U DNA polymerase is a novel engineered high fidelity enzyme developed using fusion technology.

Thermo Scientific Phusion U DNA Polymerase is a novel engineered high fidelity enzyme developed using fusion technology. Due to a proprietary mutation in the so called dUTP binding pocket of Phusion polymerase, Phusion U polymerase overcomes an important limitation of proofreading enzymes: it is able to incorporate dUTP and read through uracil present in DNA templates.

In addition to the uracil usage possibility, Phusion U polymerase features all the superior properties of other Phusion DNA polymerases: great accuracy, speed, ability to amplify long amplicons up to 20 kb, and a high specificity with Affibody-based hot start. These features make Phusion U Hot Start DNA Polymerase an excellent choice for such important applications as amplification of bisulphite-converted or damaged DNA as well as use of carryover contamination control.

Phusion U Hot Start Green DNA Polymerase is a combination of Phusion U Hot Start DNA Polymerase and 5X Phusion Green buffers. The buffers include a density reagent and two tracking dyes for direct loading of PCR products on a gel. The colored buffer does not interfere with Phusion U polymerase performance and is compatible with downstream applications such as DNA sequencing, ligation, and restriction digestion.

Highlights

  • Accuracy—high fidelity DNA polymerase (25X Taq)
  • Uracil-tolerance—engineered to incorporate dUTP and amplify uracil containing templates
  • Specificity—hot start for reduced nonspecific amplification and primer degradation
  • Speed—short extension times (15–30 s/kb)
  • Green format—permits direct loading of PCR products on gels

Applications

  • Amplification of bisulphite-converted DNA
  • Amplification of damaged or aged DNA
  • Carry-over contamination control
  • Uracil-excision based (USER) cloning methods

Using Phusion DNA polymerases

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

Specifications

Concentration 2 U/μL
Content And Storage • Phusion U Hot Start DNA Polymerase, 2 U/μL
• 5X Phusion HF & GC Buffers
• DMSO
• 50 mM MgCl2 solution

Phusion HF Buffer and Phusion GC Buffers each provide 1.5 mM MgCl2 in the final 1X concentration.
Store at -20°C.
GC-Rich PCR Performance High
Polymerase Phusion U Hot Start DNA Polymerase
Reaction Speed Fast
Product Type Hot Start DNA Polymerase
Quantity 100 units
Shipping Condition Dry Ice
For Use With (Application) Hot-start PCR, High-fidelity PCR
Fidelity (vs. Taq) 25X
Hot Start Built-In Hot Start
No. of Reactions 100 Reactions
Overhang Blunt
Reaction Format Standalone
Size (Final Product) 20 kb or less
Color Colorless
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Frequently Asked Questions (FAQs)

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.
What nucleotide analogues can I use with DyNAzyme and Phusion DNA Polymerases?

DyNAzyme II DNA Polymerase can use dUTP, biotinylated dNTPs, 7-deaza-dGTP, digoxigenin-dUTP, bromo-dUTP, radiolabeled dNTPs and ITP. DyNAzyme EXT DNA Polymerase and Phusion DNA Polymerase cannot read dUTP-derivatives or dITP in the template strand so the use of these analogues is not recommended. Use Phusion U Hot Start DNA Polymerase for amplification of dUTP and dITP containing templates.

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