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Thermo Scientific™ Phusion U Hot Start DNA Polymerases
Description
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
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 | 500 units |
| Shipping Condition | Dry Ice |
| For Use With (Application) | Hot-start PCR, High-fidelity PCR |
| Fidelity (vs. Taq) | 25X |
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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.
For Research Use Only. Not for use in diagnostic procedures.