What is Phusion polymerase used for?
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.
Why am I getting smearing after PCR?
Smearing appears due to contamination in DNA in some cases. Minimize the annealing temperature to overcome the problem.
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.
What causes smears in gel electrophoresis?
Gel electrophoresis allows scientists to visualize digested samples and measure the sizes of the fragments. Smearing results from improperly prepared agarose gels, loading an undiluted sample into the wells or using poor quality samples.
What is Phusion DNA Polymerase?
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 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.
How do you get rid of a smear in PCR?
“If the PCR products appear as a smear, you may need to increase the annealing temperature or decrease the magnesium (if you added Mg yourself and it wasn’t already in the mix.). You may also be adding too much template.” I would suggest, using freshly made gels and running in freshly prepared running buffer.
Why DMSO is added in PCR reaction?
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 does smearing on a gel mean?
If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear. If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel.
What is a DNA smear?
A smear along the path of nucleic acid movement is simply many bands that cannot be easily distinguished. For example, sizes of molecules in genomic DNA or degraded RNA vary extensively so they are manifested as smears.
Is Phusion High-Fidelity?
5 days ago
Thermo Scientific Phusion High-Fidelity DNA Polymerases set a gold standard for high performance PCR.
Is Phusion a Taq polymerase?
What is DNA smear?
Does DMSO denature RNA?
Indeed, high DMSO concentrations (>75%) have previously been shown to disrupt the structure and stability of RNA27,28 and DNA.
What is a Phusion ® DNA polymerase?
Phusion ® DNA Polymerase exploits this dramatic increase in processivity, resulting in shorter extension times, more robust and high yield amplification, and the ability to amplify long templates in a fraction of the time. Click here to acc ess our Tm calculator. Phusion ® was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific.
What is the correct buffer for my DNA polymerase?
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. Each of these buffers contains MgCl 2 (1.5 mM at the final [1X] reaction concentration).
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 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.