What does Phusion DNA 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 do you understand with high fidelity and thermostable DNA Polymerase?
High-fidelity DNA polymerases have several safeguards to protect against both making and propagating mistakes while copying DNA. Such enzymes have a significant binding preference for the correct versus the incorrect nucleoside triphosphate during polymerization.
Does phusion have exonuclease activity?
Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products.
What is Phusion Flash?
Phusion Flash II DNA Polymerase is a proofreading polymerase that contains a unique processivity- enhancing domain, making this polymerase accurate and rapid. Phusion Flash II DNA Polymerase is a hot start polymerase utilizing a reversibly binding Affibody® protein.
Why is Taq used over Pfu?
The downside of Pfu is its speed which is slower that that of Taq. Combining Pfu and Taq gives you the best option as you get the speed of Taq with the fidelity of Pfu.
What is thermostable polymerase?
The thermostable DNA polymerase is perhaps the most important target site of PCR-inhibiting substances. The most widely used polymerase in PCR-based methods for the detection of microorganisms is Taq DNA polymerase from Thermus aquaticus.
Which thermostable enzymes have high fidelity?
Several thermostable DNA polymerases with 3′→5′ exonuclease-dependent proofreading activity ( Pfu, Vent, Deep Vent and UlTma ) have been introduced for high fidelity PCR amplification ( 1–3 ).
How is Pfu different from Taq?
Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3′ to 5′ exonuclease proof reading activity, meaning that it works its way along the DNA from the 5′ endto the 3′ endand corrects nucleotidemisin corporation errors. Pfu DNA polymerase-generated PCRfragments will have fewer errors than Taq-generated PCR inserts.
Why are vent and Pfu polymerases more efficient than Taq polymerases?
7. Why are vent polymerase and Pfu more efficient than the Taq polymerase? Sol:(a) Because of proofreading activity.
Why thermostable DNA polymerase is used in PCR?
T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. Therefore, it replaced the DNA polymerase from E. coli originally used in PCR.
What does DMSO do to DNA?
DMSO binds to the DNA and prevents the reannealing of single-stranded DNA. It also facilitates the annealing of primer with a templet. Therefore, it increases the specificity and yield of the PCR reaction.
Does DMSO inhibit PCR?
DMSO is used in PCR to inhibit secondary structures in the DNA template or the DNA primers. It is added to the PCR mix before reacting, where it interferes with the self-complementarity of the DNA, minimizing interfering reactions. DMSO in a PCR reaction is applicable with high GC-content(to decrease thermostability).
Which is a thermostable DNA polymerase?
The thermostable DNA polymerase most commonly used is taq DNA polymerase, isolated from the thermophilic bacterium Thermus aquaticus, which was discovered growing in hot springs at 75°C at Yellowstone National Park.
What is the best way to dilute Phusion DNA polymerase?
Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors. Please note that protocols with Phusion DNA Polymerase may differ from protocols with other standard polymerases. As such, conditions recommended below should be used for optimal performance. Component.
What is the optimal concentration of Phusion DNA polymerase?
However, the optimal concentration of Phusion DNA Polymerase may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 µl reaction, especially for amplicons longer than 5 kb. 5X Phusion HF Buffer and 5X Phusion GC Buffer are provided with the enzyme.
What is Thermo Scientific Phusion high-fidelity DNA polymerase?
Manufactured and quality-controlled at New England Biolabs, Thermo Scientific ® Phusion High-Fidelity DNA Polymerase offers both high fidelity and robust performance. High-Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification.
What is the final concentration of dNTP in a DNA polymerase?
Phusion DNA polymerase is also compatible with other additives such as formamide or glycerol. The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Phusion cannot incorporate dUTP.