What is a long range PCR?
Long range PCR refers to the amplification of DNA targets over 5kb in length which typically cannot be amplified using routine PCR methods or reagents. Whether for cloning, genome mapping or sequencing, PCR Biosystems has a range of solutions enabling the amplification of long fragments with speed and high fidelity.
How long can PCR amplicons be?
For standard PCR scientists generally design amplicons to be between 200–1000 bp. For quantitative PCR, standard amplicons range from 75–150 bp. It is unlikely that an amplicon will be too short.
What is Phusion polymerase used for?
The Phusion™ High–Fidelity DNA Polymerase brings together a Pyrococcus–like enzyme with a processivity–enhancing domain. The Phusion™ High– Fidelity DNA Polymerase generates long amplicons with accuracy and speed, even on the most difficult templates.
Is Phusion High Fidelity DNA polymerase?
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 are PCR long products?
Long and accurate polymerase chain reaction (LA PCR) refers to the production of amplified product longer than ∼3 kilobases (kb) with high fidelity. Long PCR mixtures typically yield PCR products with some tenfold fewer mutations1 than those observed in products resulting from conventional PCR.
How do you optimize PCR conditions?
PCR conditions
- Use higher denaturation temperatures (e.g., 98°C as opposed to 94°C or 95°C) to allow complete denaturation of the template.
- Keep annealing times for GC-rich templates as short as possible.
- Use primers with a higher Tm (>68°C), because annealing can occur at a higher temperature.
How long should PCR products be?
How long can you keep PCR product?
You can store PCR product till one week in Room Temperature, For one or two month kept in 4 and for longer till one 1 year store at -20 degree centigrade.
What is Phusion PCR?
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.
How does Phusion polymerase work?
Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products. A 3.8 kb fragment was amplified from 50 ng of Jurkat gDNA using different polymerases. Reactions were carried out according to the manufacturer’s recommended conditions.
What is Phusion 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 many reactions can be performed in Phusion high fidelity PCR kit?
The Phusion High-Fidelity PCR Kit contains a sufficient supply of Phusion High-Fidelity DNA Polymerase, Phusion HF and GC Buffers, deoxynucleotides, magnesium chloride, DMSO, and DNA size standard to perform 50 reactions (small) or 200 reactions (large). Control template and primers are provided for 20 control reactions.
What is the extension time for Phusion flash PCR master mix?
We recommend extension times of 15 s/kb for Phusion Flash PCR Master Mix. 15 s/kb is a conservative value that we can promise to work with almost any amplicon. In many cases, significantly shorter extension times (0-5 s/kb) can be used without compromising the yield.
When to use Phusion high fidelity DNA polymerase?
High-Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification. Phusion High-Fidelity DNA Polymerase offers both high-fidelity and robust performance, and thus can be used for all PCR applifications.
Which is the best thermocycling protocol for Phusion?
Generally, 25–35 cycles yields sufficient product. When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol is recommended. The PCR products generated using Phusion DNA Polymerase have blunt ends; if cloning is the next step, then blunt-end cloning is recommended.