Dna polymerase is capable of adding nucleotides to the free. Dna polymerase and 18fold lower than taq dna polymerase, making it the highest fidelity enzyme available see table i. Phusion highfidelity pcr master mix is convenient 2x mix containing phusion dna polymerase, nucleotides and optimized reaction buffer including mgcl. Difference between phusion and taq polymerase compare. The polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification. Amplification of a 500bp fragment using pfuultra highfidelity. Datasheet for phusion highfidelity dna polymerase neb. Phusion dna polymerase is supplied with stan dard 5x phusion. Phusion highfidelity dna polymerase high performance for.
Due to their high accuracy, robustness, reliability and inhibitor tolerance, phusion highfidelity dna polymerases provide outstanding performance for highfidelity pcr. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. Phusion highfidelity dna polymerase thermo fisher scientific. These results suggest that of the six commerciallyavailable dna polymerases we tested, kod.
Protocols optimized for phusion dna polymerase can. While most may think standard taq is the backbone of pcr, there are many other dna polymerase options out there. They are the key polymerizing enzymes that involve during replication. Phusion highfidelity dna polymerases product listing product overview 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, new england biolabs. At the top of the page youll find a map of all positions of known mutants. Component amount storage phusion hot start ii dna polymerase, 2 u.
Jul 27, 2017 ms analysis of the hairpin modification h4 by the phusion and pfu polymerases at 64 c for 2 hours in the presence of 200. For routine pcr under 7 kb, phusion is an absolute workhorse. Phusion hot start dna polymerase tends to work better at elevated denaturation and. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. Phusion highfidelity dna polymerase new england biolabs. Dna polymerases are widely used enzymes in molecular biology techniques and are also naturally present in all organisms that undergo dna replication. Phusion the wolfson centre for applied structural biology. Protocol for a routine pcr with phusion highfidelity pcr kit.
Pcr protocol for phusion highfidelity dna polymerase. Phusion highfidelity dna polymerases have become the choice for even. New insights into the quikchangetm process guide the use. Kod polymerase novagen pdf merck novagen kod dna polymerase kod dna polymerase dna polymerases pcr reagents and kits. 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. Three types of rna polymerase in eukaryotic nuclei type location rna synthesized effect of. All components should be mixed and centrifuged prior to use. The polymerase you use has a significant impact on the. Thermo scientific phusion hot start ii highfidelity dna polymerase. Phusion offers several advantages over pfuturbo, being faster, more robust and with higher fidelity. L reaction volume gives good results, but the optimal amount can range from 0. New insights into the quikchangetm process guide the use of. Rapid protocol times can be achieved with phusion flash highfidelity pcr master mix, a product developed especially for fast pcr.
Mar 24, 2020 merck novagen kod dna polymerase kod dna polymerase dna polymerases pcr reagents and kits. 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. Add phusion last, minimize time out of freezer program pcr machine before adding phusion add each component in order listed above to a pcr tube note. Pcr using phusion high fidelity dna polymerase biocompare. Pcr with highfidelity dna polymerase igem 2018 projects. In the laboratory setting, pfu is used to amplify dna in the polymerase chain reaction pcr, where the enzyme serves the central function of copying a new strand of dna during each.
The phusion high fidelity dna polymerase possesses the following activities. Phusion dna polymerase is a very high fidelity polymerase which is artificially constructed by dna binding domain to proofreading polymerase from pyrococcus furiosus. L reaction depending on the amplicon length and difficulty. Phusion dna polymerases incorporate more nucleotides per binding event as compared to other polymerases.
Thermo scientific phusion rtpcr kit product information. This is the pcr protocol for phusion highfidelity dna polymerase m0530. Highfidelity dna polymerases are important for applications in which the dna sequence needs to be correct after amplification. Incorporating an exciting new technology, phusion dna polymerase brings. A single unit of phusion dna polymerase produced higher yields than 2.
Phusion dna polymerase may be diluted in 1x hf or gc buffer just prior to use in order to reduce pipetting errors. The phusion sitedirected mutagenesis kit contains reagents for a total of 20 mutagenesis reactions including control reactions, and control plasmid and primers for 10 reactions. Due to their high accuracy, robustness, reliability and. Q5 dna polymerase is composed of a novel polymerase that is fused to the processivityenhancing sso7d dna binding domain, improving speed, fidelity and reliability of performance. Phusion hot start dna polymerase ensures high fidelity for the exponential. Apr 19, 2020 finnzymes phusion highfidelity dna polymerase offers extreme performance for all pcr applications. I have used phusion high fidelity polymerase for amplification of dna templatesgenes needed for. Phusion dna polymerases thermo fisher scientific us. 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. The phusion dna polymerase generates long templates with an accuracy and speed previously. Rna preparation may be removed by rnasefree dnase i treatment. Incorporating an exciting new technology, phusion dna polymerase brings together a novel pyrococcuslike enzyme with a processivityenhancing domain. In the pcr reaction, phusion dna polymerase extends the primers.
Phusion dna polymerases produce blunt end dna products. Phusion sitedirected mutagenesis kit user guide pub. Pfu dna polymerase is an enzyme found in the hyperthermophilic archaeon pyrococcus furiosus, where it functions to copy the organisms dna during cell division. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult gcrich amplicons. Massspectrometry analysis of modifications at dna termini. Request a sample of thermo scientific phusion dna polymerase. Phusion hot start flex dna polymerase is available as standalone enzyme or in a master mix format and enables high specificity amplification of a broad range of templates. Shop online for a wide selection of emd millipore novagen kod hot start dna polymerase for pcr amplification of long strand and gcrich dna templates. This should be done especially if primers for the pcr step cannot. Guidelines for using phusion dna polymerase ng to ensure homogeneity and improve recovery. Jun 18, 2015 taq polymerase was named after the thermophilic bacterium thermus aquaticus from which it was isolated in 1965 by thomas d. 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.
Q5 highfidelity dna polymerase new england biolabs. It generates blunt ends in the amplification products. Usually 1 unit of phusion hot start ii dna polymerase per 50. The unique composition of phusion flash highfidelity pcr master mix enables the use of extremely short pcr protocols 15 s1 kb with both low and high complexity dna templates. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification.
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. We generally recommend using phusion dna polymerase at a concentration of 20 unitsml 1. Difference between phusion and taq polymerase compare the. Protocol for a routine pcr with phusion highfidelity pcr. Hemo klentaq dna polymerase was resistant to blood components at concentrations up to 20%, while mighty amp, phusion blood ii, and kapa blood dna. Jul 06, 2019 finnzymes phusion highfidelity dna polymerase offers extreme performance for all pcr applications. This page presents all the information in polbase for phusion. You can get yourself a free sample to test it if you have problems with phusion or any other polymerase. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. Wang department of pathology stanford university school of medicine stanford, california 943055324 enzymatic properties and characteristics that distinguish each dna polymerase during the past decade, five dna polymerases pol have been charac terized in eukaryotic cells. However, the optimal concentration of phusion dna polymerase may vary from 1040 unitsml 0. 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. Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol.
It is important to add phusion dna polymerase last in order to prevent any primer degradation caused by the 3. Overlap extension pcr, phusion polymerase or taq polymerase. Wang department of pathology stanford university school of medicine stanford, california 943055324 enzymatic properties and. However, due to the novel nature of phusion hot start dna polymerase, optimal reaction conditions may differ from standard enzyme protocols. Learn more about phusion highfidelity dna polymerase. Pcr amplifies specific dna sequences exponentially by using multiple cycles of a threestep process. While diyphusion wont replace commercial q5 in our lab, it allows us to save it for when such a high fidelity enzyme is called for. Phusion highfidelity dna polymerase high performance for all. Amplification of a 500bp fragment using pfuultra highfidelity dna polymerase results in errors in less than 0. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. Reactions can also be optimized using the provided dmso or mgcl 2 solutions. Prepare a master mix for the appropriate number of samples to be amplified. Thermocycling conditions were standard as recommended in the protocol except for extension time. Phusion dna polymerase should be pipetted carefully and gently as.
Polymerase pcr protocol for phusion highfidelity dna. Thermo scientific phusion flash highfidelity pcr master mix is a 2x master mix based on modified phusion hot start ii dna polymerase. Oct 19, 2012 hemo klentaq dna polymerase was resistant to blood components at concentrations up to 20%, while mighty amp, phusion blood ii, and kapa blood dna polymerases were resistant to blood components at concentrations up to 10% of the reaction mixture fig. The optimal amount of enzyme depends on the amount of template and the length of the pcr product. Finnzymes phusion highfidelity dna polymerase offers extreme performance for all pcr applications. Phusion dna polymerase gave strong specific bands even with the shortest extension time, completing the 3. We enable science by offering product choice, services, process excellence and our people make it. Enzyme amounts also indicate that significantly less of the highly processive phusion dna polymerase is required to complete the task. Both new england biolabs and thermo fisher scientific offer phusion.
Datasheet for phusion highfidelity dna polymerase m0530. Phusion dna polymerase is supplied with standard 5x. This high processivity allows extremely short extension times and consequently reduced protocol times. First, the doublestranded dna template is denatured at a high temperature. Protocols optimized for phusion dna polymerase can directly be applied to phusion hot start dna polymerase reactions. The speciality of phusion is that it is fused to a dnabinding proteins domain thats why its name is phusion, so that it stays attached to the template dna over a longer distance than usual. Get a complimentary sample of thermo scientific phusion highfidelity dna polymerase when you fill out the form on this page.