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?
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 |
What type of polymerase is phusion?
Fidelity DNA Polymerase
GoDirect:
| Hot Start | No |
|---|---|
| Polymerase | Phusion High Fidelity DNA Polymerase |
| Product Line | Phusion |
| Product Type Specs | High-Fidelity DNA Polymerase |
| Reaction Format | Standalone |
What is the Phusion protocol for DNA polymerase?
Protocol Component 20 µl Reaction 50 µl Reaction Final Concentration Nuclease-free water to 20 µl to 50 µl 5X Phusion HF or GC Buffer 4 µl 10 µl 1X 10 mM dNTPs 0.4 µl 1 µl 200 µM 10 µM Forward Primer 1 µl 2.5 µl 0.5 µM
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.
When to add Phusion to the PCR protocol?
It is important to add Phusion DNA Polymerase last in order to prevent any primer degradation caused by the 3´→ 5´ exonuclease activity. Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors.
What is the error rate of Phusion high fidelity DNA polymerase?
(see reference 1 on page 4), the error rate of Phusion™ High–Fidelity DNA Polymerase in Phusion™ HF Buffer is determined to be 4.4 × 10-7, which is approximately 50 fold lower than that of the Thermus aquaticus DNA polymerase, and 6-fold lower than that of the Pyrococcus furiosus DNA polymerase.
