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What does Phusion polymerase do?

What does Phusion polymerase do?

Phusion DNA Polymerase brings together a novel Pyrococcus-like enzyme with a processivity-enhancing domain and generates PCR products with accuracy and speed previously unattainable with a single enzyme, even on your most difficult templates. Additionally, Phusion DNA Polymerase is capable of amplifying long templates.

What is the optimal running temperature of Phusion polymerase?


Initial Denaturation 98°C 30 seconds
25-35 Cycles 98°C 45-72°C 72°C 5-10 seconds 10-30 seconds 15-30 seconds per kb
Final Extension 72°C 5-10 minutes
Hold 4-10°C

What is Phusion High-Fidelity DNA Polymerase?

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

What is the difference between phusion and Taq polymerase?

Phusion is more highly processive than Taq, so it’s faster than Taq and can handle much longer amplicons, because it has a “clamp” that keeps it attached to the DNA strand where as Taq is more likely to dissociate. Commercially purified Taq polymerase doesn’t have a proof-reading domain, so it has a higher error rate.

What is in Phusion HF buffer?

The 5X Phusion HF Buffer contains 7.5 mM MgCl2, which provides 1.5 mM MgCl2 in final reaction conditions. The 5X Phusion GC Buffer contains 7.5 mM MgCl2, which provides 1.5 mM MgCl2 in final reaction conditions. Both Phusion Buffers supply 1.5 mM MgCl2 at final reaction conditions.

What is in GC buffer?

It is a 2X master mix consisting of Phusion DNA Polymerase, deoxynucleotides and reaction buffer that has been optimized and includes MgCl2. All that is required is the addition of template, primers and water.

Is Phusion a Taq polymerase?

Phusion DNA Polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. With an error rate > 50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase (1), Phusion is one of the most accurate thermostable polymerases available.

What makes Taq polymerase unique?

Taq DNA polymerase is the most common enzyme used for PCR amplification. This enzyme is extremely heat resistant with a half-life of 40 minutes at 95°C. This will result in the amplification of non-specific targets that can be overcome by the use of a ‘hot-start’ PCR technique (Mullis 1991).

What does 5X Phusion buffer do?

5X Phusion Green Buffers include a density reagent and two tracking dyes for direct loading of PCR products on a gel. The colored buffers do not interfere with the enzyme performance and are compatible with downstream applications such as DNA sequencing, ligation and restriction digestion.

What would happen without DNA polymerase?

Without the copying of the DNA life would not continue as existing organisms would not be able to reproduce and replace themselves. Life is dependent on the information stored on the DNA. Without replication of the DNA the information would not be passed on and life would cease to exist.

Who is the manufacturer of Phusion DNA polymerase?

Phusion DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific. PHUSION ® is a registered trademark of Thermo Fisher Scientific.

How many units of Phusion DNA polymerase should I use?

The optimal amount of enzyme depends on the amount of template and the length of the PCR product. Usually 1 unit of Phusion DNA Polymerase per 50 μL reaction volume gives good results, but the optimal amount can range from 0.5 to 2 units per 50 μL reaction depending on amplicon length and difficulty. It is not recommended to exceed 2 U/50 μL

Why does Phusion HF DNA polymerase fail to amplify?

Phusion HF DNA polymerase fails to amplify whereas Taq DNA polymerase succeeds. What could be the reason for this? I am trying to PCR amplify a 4kb gene sequence from a vector.

What is the TM of Phusion HiFi polymerase?

The Tm of my primer pair using Phusion HiFi polymerase is 66 degrees C, as calculated by the NEB Tm calculator. One primer is 20 nt long and the other is 22 nt long with very similar GC content. I have tried using both circular and linearized plasmid as template at a final mass of ~15-20 ng in a 50 uL reaction volume.