Id like to clone a fragment amplified with phusion highfidelity dna polymerase. Get a complimentary sample of thermo scientific phusion highfidelity dna polymerase when you fill out the form on this page. So i did the reaction with phusion at 70 degrees, and then at 63 when the first one didnt work. Because my primers are so long, they have relatively high tms. Phusion highfidelity pcr master mix with hf buffer. Its unique structure, a novel pyrococcuslike enzyme fused with a processivityenhancing domain, increases fidelity and speed. 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 gcrich templates. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. This protocols is for pcr using q5 highfidelity dna polymerase m0491. Phusion the wolfson centre for applied structural biology.
Pcr protocol for phusion highfidelity dna polymerase. Thermo scientific phusion rtpcr kit product information. Protocol pcr the polymerase chain reaction polymerase chain reaction mullis, k. According to the neb gibson planner, after inputting that i will be using phusion, they told me to use 72 degrees as my tm. Thermo scientific phusion highfidelity pcr master mix. A variant of this protein with an additional 65 amino acid changes is sold as phusion polymerase by new england biolabs. Phusion sitedirected mutagenesis kit user guide pub. This protocol relies on phusion dna polymerase, which is an ideal choice for pcr applications using samples of variable dna quality. The polymerase chain reaction pcr, first envisaged in 1984 by kary mullis, has revolutionized life sciences and has become an essential technique in many aspects of science, including clinical diagnostics, forensics and genetic engineering. Phusion hot start flex dna polymerase is supplied with standard 5x phusion hf buffer, as well as 5x phusion gc buffer, which can be used for complex or gcrich templates. Taking the human whole blood as template, a target fragment of 1,295 bp was amplified with phanta max superfidelity dna polymerase, a high fidelity dna polymerase from company a, and a high fidelity dna polymerase. If tacloning is preferred, then dna should be purified prior to aaddition, as phusion dna polymerase.
Due to the nature of the phusion highfidelity dna polymerase. It eliminates the need for a separate dna purification step prior to pcr. L phusion highfidelity dna polymerase, 2x phusion hf buffer in f531 or 2x phusion gc buffer in f532, and 400. Phusion highfidelity pcr master mix with hf buffer is a 2x master mix consisting of phusion dna polymerase, deoxynucleotides and reaction buffer that has been optimized and includes. It generates blunt ends in the amplification products.
Phusion highfidelity pcr master mix with hf buffer is a 2x master mix consisting of phusion dna polymerase, deoxynucleotides and reaction buffer that has been optimized and includes mgcl 2. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. Request a sample of thermo scientific phusion dna polymerase. I have been trying to introduce an insertion into my gene of interest following a protocol similar to that of the quikchange lightning. Thermo scientific phusion highfidelity dna polymerase. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Both phusion hf buffer and phusion gc buffer provide 1. Protocols optimized for phusion dna polymerase can directly be applied to phusion hot start dna polymerase reactions. Phusion hot start flex dna polymerase is unlike other enzymes and care must be taken when designing cycling protocols. Phusion dna polymerases thermo fisher scientific us. For the subsequent amplification of the cdna template. Pcr from ffpe tissues without dna extraction using phusion. Phusion hot start flex dna polymerase is supplied with standard 5x phusion hf buffer, as well as 5x phusion.
The following guidelines are provided to ensure successful pcr using phusion dna polymerase. Dna polymerase 1, phusion is one of the most accurate. Thermostable phusion dna polymerase is isolated and purified from an e. Phusion hot start flex dna polymerase new england biolabs. With the phusion sitedirected mutagenesis kit, point mutations, insertions and deletions can be introduced in any type of plasmid dna. Phusion highfidelity dna polymerase, new england biolabs. Sep 20, 2017 phusion dna polymerase is a very high fidelity polymerase which is artificially constructed by dna binding domain to proofreading polymerase from pyrococcus furiosus. The modified phusion hot start ii highfidelity dna polymerase. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. Make sure to keep the enzymes and dntp stocks on ice when taken outside the. Difference between phusion and taq polymerase compare the. Phusion highfidelity dna polymerase high performance for all.
Phusion dna polymerases tend to work better at elevated denaturation and annealing temperatures due to. Make sure to keep the enzymes and dntp stocks on ice when taken outside the freezer. Thermo scientific phusion green highfidelity dna polymerase 2 ul 100u. Using, pfu dna polymerase in your pcr r eaction results in a blunt ended pcr products, which are ideal. Due to the unique nature of phusion dna polymerases, please pay special attention to. Overlap extension pcr, phusion polymerase or taq polymerase.
As a result of this unique functional pairing, phusion dna polymerases are capable of generating pcr products with very high accuracy and speed. Product information thermo scientific phusion site. This kit uses the highly processive phusion hot start ii dna polymerase for exponential pcr amplification of dsdna plasmid to be mutated. The addition of an aptamerbased inhibitor allows room temperature reaction setup. Due to their high accuracy, robustness, reliability and inhibitor tolerance, phusion highfidelity dna polymerases provide outstanding performance for highfidelity pcr. New insights into the quikchangetm process guide the use of. According to the new understanding, we developed a new protocol with phusion dna polymerase and partially overlapping primers to generate the pcr products for efficient sitedirected mutagenesis. Polymerase, the optimal reaction conditions may differ from. Phusion hot start flex dna polymerase offers robust, high fidelity performance and room temperature reaction setup. This is the pcr protocol for phusion highfidelity dna polymerase m0530. Phusion highfidelity dna polymerase new england biolabs. In addition to high accuracy, pfuultra highfidelity dna polymerase provides robust amplification of long, complex genomic targets. Datasheet for phusion highfidelity dna polymerase neb. To determine the optimal annealing temperatures for a given set.
With the thermo scientific phusion sitedirected mutagenesis kit, point mutations, insertions and deletions can be introduced in any type of plasmid dna. In the quality control tests of phusion dna polymerase, 7. In agilents pfuultra ii fusion hs dna polymerase, we couple the fusion polymerase technology with our engineered pfuultra dna polymerase, hotstart antibody, and proprietary archaemaxx. Since their introduction, thermo scientific phusion highfidelity dna polymerases have been referenced in thousands of publications for highperformance pcr and have become the. Pfuultra ii fusion hs dna polymerase technical services.
The unique structure of the enzyme enables short protocol. Manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr applications. Phusion highfidelity dna polymerase thermo fisher scientific. Thermo scientific phusion u multiplex pcr master mix. Phusion highfidelity pcr master mix is convenient 2x mix containing phusion dna polymerase, nucleotides and optimized reaction buffer including mgcl. This simplifies the cdna synthesis protocol as it renders a separate rnase treatment step unnecessary. Phusion u multiplex pcr master mix, it is not necessary to perform the pcr setup on ice. However, due to the novel nature of phusion hot start dna polymerase, optimal reaction conditions may differ from standard enzyme protocols. It is ideal for assembling gblock fragments or introducing point mutations. The phusion highfidelity dna polymerase sets a new standard for pcr performance. Amplification of templates with high gc content, high secondary structure, low template concentrations or long amplicons may require further. It catalyzes the polymerization of nucleotides into duplex dna in the 5. The pcr products generated using phusion dna polymerase have blunt ends. A testing various dna polymerases for the amplification of a 1 kb fragment with 70% gccontent.
Phusion hot start ii dna polymerase does not require a separate activation step in the pcr protocol as it is immediately reactivated at high temperatures. The phusion highfidelity dna polymerase should be pipetted carefully and gently as the high glycerol content 50% in the storage buffer may otherwise lead to pipetting errors. This kit uses the highly processive phusion hot start highfidelity dna polymerase for exponential pcr ampli. Phusion blood direct pcr kit thermo fisher scientific. Im planning to use overlap extension pcr for dna insertion into a vector, as it is explained by briskin and matsmura in. The unique structure and characteristics of phusion dna polymerase make it a superior choice for cloning. Thermo scientific phusion highfidelity pcr kit life. At the top of the page youll find a map of all positions of known mutants. The five quality features of q5 high fidelity dna polymerase 1. This disclosure provides, among other things, a composition comprising.
This kit uses the highly processive thermo scientific phusion hot start ii highfidelity dna polymerase. If available, a representative structure is displayed at the. Pmc images search for phusion highfidelity dna polymerase figure 1 engineered proofreading dna polymerases have good performance. Phusion highfidelity dna polymerase for high speed and high fidelity pcr. This kit uses the highly processive phusion hot start ii dna polymerase. Will phusion highfidelity dna polymerase incorporate dutps. Phusion dna polymerase possesses the following activities. The mutagenesis protocol comprises only three steps. Similar results were obtained comparing pfux7 to phusion pfus7 polymerase see additional file 2.
Phusion polymerase protocol fermentas highfidelity pcr enzymes are used for applications requiring high accuracy during dna amplification such as cloning, sequencing or mutagenesis. Amplification of a short dna stretch by repeated cycles of in vitro dna polymerization. Illumina barcoded pairedend capture library preparation using nonindex adaptors and phusion dna polymerase. Read the quikchange manual and follow the protocol. When compared to taq dna polymerase, phusion dna polymerase. Understand the principles of the polymerase chain reaction. Phusion highfidelity dna polymerase is supplied with 5x phusion hf buffer, 5x phusion gc buffer, dmso and 50 mm mgcl2 solution. However, the optimal concentration of phusion dna polymerase may vary from 1040 unitsml 0. Phusion dna polymerases produce bluntend dna products. Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. 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. Phusion dna polymerase is supplied with standard 5x phusion hf buffer, as well as 5x phusion. The phusion highfidelity pcr kit contains a sufficient supply of phusion highfidelity dna polymerase, phusion hf and gc buffers, deoxynucleotides, magnesium chloride, dmso, and.
Pfu dna polymerase is a thermostable enzyme that replicates dna at 75c. This page presents all the information in polbase for phusion. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification. Phusion highfidelity dna polymerases choosing the right phusion product phusion highfidelity.
The following file naming structure is used to name the majority of these document files. This means that pfu dna polymerase generated pcr fragments will have fewer errors than taq generated pcr inserts. Assembling gblock fragments a 2530bp overlap with a gc content of roughly 50% between the two fragments is. A method for copying a dna template, as well as a kit for performing the same, are also described. A variant of this protein with an additional 65 amino acid changes is sold as phusion polymerase. Specific synthesis of dna in vitro via a polymerase catalysed chain reaction.
Protocol for a routine pcr with phusion highfidelity pcr. This pfu variant has the sso7d processivityenhancing domain attached that increases its. Jan 30, 2015 new insights into the quikchange tm process guide the use of phusion dna polymerase for sitedirected mutagenesis yongzhen xia, 1 wenqiao chu, 1 qingsheng qi, 1 and luying xun 1, 2, 1 state key laboratory of microbial technology, shandong university, jinan 250100, p. Phusion highfidelity pcr master mix is convenient 2x mix containing phusion dna polymerase. Highest fidelity dna amplification available at 280x higher than taq, q5 offers unparalleled fidelity for your most important samples, but with a protocol. The twostep protocol should be used when the annealing temperatures of the primers are.
A key component of pfuultra highfidelity dna polymerase is the archaemaxx. Our lab dntp stocks contain 10 mm each of datp, dttp, dctp, and dgtp. Datasheet for phusion hot start flex dna polymerase. This protocol is for expressing and purifying the pfusso7d polymerase from e. Phusion highfidelity pcr master mix with gc buffer. Next, aliquots of the reactions were treated with dpni and the dpnitreated. All that is required is the addition of template, primers and water. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard. Can i use phusion polymerase in quikchange protocol. Includes phusion dna polymerase 2 ul 5x phusion hf buffer 5x phusion gc buffer dmso 50 mm mgcl 2 solution both phusion hf buffer and phusion gc buffer provide 1. However, the optimal concentration of phusion dna polymerase.
Amplification of templates with high gc content, high secondary structure, low template concentrations or long amplicons may require further optimization. Hot start ii dna polymerase is equal to that of phusion dna polymerase 4. Why are there low molecular weight discrete bands on an agarose gel after a pcr using phusion highfidelity dna polymerase. Thermo scientific phusion blood direct pcr kit is developed for amplification of dna from whole blood. Pcr protocol for phusion highfidelity dna polymerase m0530 overview the following guidelines are provided to ensure successful pcr using phusion dna 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. Thermo scientific phusion sitedirected mutagenesis kit, point mutations, insertions and deletions can be introduced in any type of plasmid dna. Due to the unique nature of phusion u dna polymerase, optimal reaction conditions may differ from standard enzyme protocols. Us9963687b2 fusion polymerase and method for using the same.
We generally recommend using phusion dna polymerase at a concentration of 20 unitsml 1. Text links to mutants and digestion products are listed in the mutants section. Phusion highfidelity dna polymerase high performance for. The unique phusion highfidelity dna polymerase offers robust performance and can be used for all pcr applications. This protocol provides instructions for preparing dna paired. Dna polymerase for exponential pcr amplification of dsdna plasmid to be mutated.
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