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Thermo Scientific™ Phusion™ Hot Start II DNA Polymerase & dNTP Mix (10 mM each)
Description
Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase sets a high standard for high performance PCR. Featuring an error rate 52-fold lower than that of Taq and 6-fold lower than that of Pfu, this polymerase is an excellent choice for cloning and other applications that require high fidelity and offers robust performance with short protocol times, even in the presence of PCR inhibitors. Phusion Hot Start II High-Fidelity DNA Polymerase generates higher yields with lower enzyme amounts than other DNA polymerases.
With Phusion Hot Start II High-Fidelity DNA Polymerase, 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' to 5' exonuclease activity of the polymerase, thus preventing degradation of primers and template DNA during reaction setup. At temperatures that promote polymerase activity, the ligand is released, rendering the polymerase fully active. Phusion Hot Start II High-Fidelity DNA Polymerase is immediately reactivated at high temperatures and does not require a separate activation step in PCR protocols.
Features
- High fidelity (52X Taq)
- No non-specific amplification and primer degradation during reaction setup
- Fast PCR due to short extension times (15–30 s/kb)
- Increased product yields with minimal enzyme amounts
Applications
- Standard PCR
- qPCR
- High fidelity and long PCR
- LAMP-PCR
- cDNA synthesis
- RT-PCR, RDA, MDA
- DNA labeling and sequencing
dNTP Mix
Thermo Scientific dNTP Mix (Cat. No. R0192) contains pre-mixed aqueous solutions of dATP, dCTP, dGTP, and dTTP, each at a final concentration of 10 mM. The nucleotides have greater than 99% purity and are free of nuclease activities, as well as human and E. coli DNA. Mixes offer the possibility of reducing the number of pipetting steps and the risk of reaction setup errors.
- Greater than 99% purity confirmed by HPLC
- Free of human and E. coli DNA
- Stable after multiple freeze-thaw cycles
- Up to 95% of dNTPs remain in triphosphate form after 7 weeks at room temperature
- Up to 90% of dNTPs remain in triphosphate form after 30 cycles of PCR (1 minute at 94°C; 3 minutes at 72°C)
Notes
- This package includes Phusion Hot Start II High-Fidelity DNA Polymerase (4 x 500 U) and high-quality dNTP Mix (10 mM each, 2 x 1 mL).
- 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 Hot Start II High-Fidelity DNA Polymerase (4 x 500 U at 2 U/μL) • 10 mM dNTP Mix (2 x 1 mL) • 5X Phusion HF & GC Buffers • DMSO • 50 mM MgCl2 solution Store at –5°C to –30°C. |
| Detection Method | Primer-probe |
| GC-Rich PCR Performance | High |
| Polymerase | Phusion Hot Start II DNA Polymerase |
| Reaction Speed | Fast |
| Product Type | Hot Start DNA Polymerase and dNTP Mix |
| Quantity | 2000 reactions |
| Shipping Condition | Dry Ice |
| For Use With (Application) | Hot-start PCR, High-fidelity PCR |
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Frequently Asked Questions (FAQs)
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).
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.
Yes, protocols optimized for Phusion DNA Polymerase can be applied to Phusion Hot Start II DNA Polymerase reactions.
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.
For Research Use Only. Not for use in diagnostic procedures.