What is in Phusion HF buffer?
What is in Phusion HF buffer?
5X Thermo Scientific Phusion HF Reaction Buffer is a buffer solution optimized for high-fidelity DNA amplification using Phusion High-Fidelity DNA polymerases. The buffer contains 7.5 mM MgCl2 which provides 1.5 mM MgCl2 in final reaction conditions.
How does Phusion polymerase work?
Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products. Phusion DNA Polymerase is supplied with standard 5X Phusion HF Buffer, as well as 5X Phusion GC Buffer, which can be used for complex or GC-rich templates.
What is the difference between phusion and Taq polymerase?
Phusion is a DNA polymerase isolated from Pyrococcus furiosus and is mainly used in cloning experiments to increase the fidelity. Taq DNA Polymerase is the standard DNA polymerase used in the Polymerase Chain Reaction (PCR), and it is isolated from the thermostable bacterium, Thermus aquaticus.
What is Phusion High Fidelity DNA Polymerase?
Thermo Scientific Phusion High-Fidelity DNA Polymerases set a gold standard for high performance PCR. Featuring an error rate 50-fold lower than that of Taq, and 6-fold lower than that of Pfu, Phusion High-Fidelity DNA Polymerase is excellent choice for cloning and other applications requiring high fidelity.
Where did Taq polymerase come from?
DNA polymerase I from Thermus aquaticus (Taq polymerase) is the most famous representative enzyme among the thermostable DNA polymerases. Taq polymerase was identified from T. aquaticus isolated from Yellowstone National Park in Montana, USA. The report was published by Chien et al.
How do you dilute Phusion 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.
What is the optimal running temperature of Phusion polymerase?
Protocol
| STEP | TEMP | TIME |
|---|---|---|
| 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 |
Why is phusion better than Taq?
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.
Where is Phusion polymerase derived from?
Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions to copy the organism’s DNA during cell division.
Does Phusion polymerase create blunt ends?
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.
Why is DMSO used in 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).
What temperature does Taq polymerase denature?
95 °C
2. Incubate the reaction for 12 cycles in the thermocyler using the following program: initial denaturation: 2 min at 95 °C; denaturation: 45 s at 95 °C; annealing: 45 s at 58 °C; elongation: 1 min at 72 °C; final elongation: 5 min at 72 °C (see Note 3).
https://www.youtube.com/watch?v=DM69lyeZURg