This page presents all the information in polbase for phusion. The enzyme, originally developed by finnzymes, is a fusion of a proofreading polymerase and a processivityenhancing domain, creating one of the most reliable and robust polymerases available. I was wondering if anyone has ever seen large deletions 4050 bp occur when using phusion polymerase to clone a gene. Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol. Dna polymerase for exponential pcr amplification of dsdna plasmid to be mutated. February 12, 2020 admin food leave a comment on kod polymerase novagen pdf merck novagen kod dna polymerase kod dna polymerase dna polymerases pcr reagents and kits. This product is manufactured by new england biolabs. 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. Enzymatic synthesis of modified oligonucleotides by pear. Protocols optimized for phusion dna polymerase can directly be applied to phusion hot start dna polymerase reactions. Therefore, the hf buffer should be used as the default buffer for highfidelity amplification. 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.
The following guidelines are provided to ensure successful pcr using phusion dna polymerase. Phusion dna polymerase is a superior choice for cloning and sets the. This product is manufactured by new england biolabs, inc. Choose phusion polymerase from thermo for highly accurate, fast and robust pcr.
Sudden problems with cdna pcr using phusion polymerase. According to the neb gibson planner, after inputting that i will be using phusion, they told me to use 72 degrees as my tm. Im planning to use overlap extension pcr for dna insertion into a vector, as it is explained by briskin and matsmura in. Usually 1 unit of phusion hot start ii dna polymerase per 50. A weak amplification of the 18 kb fragment was detected by using phusion and pfufly but not q5 and cobuddy fig. Phusion dna polymerase is a very high fidelity polymerase which is artificially constructed by dna binding domain to proofreading polymerase from pyrococcus furiosus. All that is required is the addition of template, primers and water. Source organism phusion is extracted from extremophilic archaea. The polymerase chain reaction polymerase chain reaction mullis, k. Systematic analysis of enzymatic dna polymerization using oligo. Among the five types of thermostable dna polymerases used. This disclosure provides, among other things, a composition comprising.
Dna polymerase and 18fold lower than taq dna polymerase, making it the highest fidelity enzyme available see table i. Text links to mutants and digestion products are listed in the mutants section. Phusion the wolfson centre for applied structural biology. 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. Request a sample of thermo scientific phusion dna polymerase. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. Phusion dna polymerase is very thermo stable and can be left out at room temp for days with no effect on activity.
However, gc buffer can improve the performance of phusion dna polymerase on some difficult or. Phusion hot start dna polymerase tends to work better at elevated denaturation and. Phusion highfidelity dna polymerase high performance for. This pfu variant has the sso7d processivityenhancing domain attached that increases its speed and processivity. However, i thought i would use the phusion hf pcr master mix i already had rather than order the quikchange kit, as i know phusion is a processive polymerase, and thought it would be able to.
I have recently tried using circular polymerase extension cloning to clone a 200 bp insert into a 6. At the top of the page youll find a map of all positions of known mutants. A variant of this protein with an additional 65 amino acid changes is sold as phusion polymerase by new england biolabs. Due to a proprietary mutation in the so called dutp binding pocket of phusion, phusion u overcomes an important limitation of proofreading enzymes it is able to incorporate dutp and read through uracil present in dna templates. In our lab, finnzymes phusion dna polymerase was bought for cloning lengthy and difficult dna strands. Get a complimentary sample of thermo scientific phusion highfidelity dna polymerase when you fill out the form on this page. 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. We enable science by offering product choice, services, process excellence and our people make it. Phusion highfidelity dna polymerase thermo fisher scientific. Phusion dna polymerase was developed by finnzymes oy, now a part of thermo fisher scientific. L reaction volume gives good results, but the optimal amount can range from 0. Thermo scientific phusion rtpcr kit product information. Thermo scientific phusion u dna polymerase is a novel engineered high fidelity enzyme developed using fusion technology. Finnzymes phusion highfidelity dna polymerase offers extreme performance for all pcr applications.
Amplification of templates with high gc content, high secondary structure, low template concentrations or long amplicons may require further optimization. Phusion highfidelity pcr master mix is convenient 2x mix containing phusion dna polymerase, nucleotides and optimized reaction buffer including mgcl. 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 extension step. Both phusion hf buffer and phusion gc buffer provide 1. 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. 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. Us9963687b2 fusion polymerase and method for using the. In the attached pdf file, you can see, that my grad student included two more samples for the phusion polymerase, where she used the same conditions, but a different.
Can i use phusion polymerase in quikchange protocol. Due to their high accuracy, robustness, reliability and inhibitor tolerance, phusion highfidelity dna polymerases provide outstanding performance for highfidelity pcr. 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. When cloning fragments amplified with phusion dna polymerases, blunt end cloning is recommended. Specific synthesis of dna in vitro via a polymerase catalysed chain reaction.
Phusion dna polymerases the best choice for highfidelity cloning. If ta cloning is required, it can be performed by adding 3 a overhangs to the blunt pcr product with a different polymerase e. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Phusion highfidelity dna polymerase is supplied with 5x phusion hf buffer, 5x phusion gc buffer, dmso and 50 mm mgcl2 solution. Here we compare the features of taq, pfu and phusion polymerases using an example of onestep assembly of an 581 bplong dna sequence. It generates blunt ends in the amplification products.
If available, a representative structure is displayed at the. Fs double modified oligonucleotides using kod dna polymerase and phusion dna polymerase. Because my primers are so long, they have relatively high tms. This kit uses the highly processive phusion hot start highfidelity. Phusion dna polymerase in phusion hf buffer is determined to be 4. Difference between phusion and taq polymerase compare. 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.
Phusion hot start ii green master mix a and proofreading hotstart dna polymerases from other suppliers b merck kod hot start, c agilent pfu ultra ii hs, d kapa biosystems kapa hifi hs, and e takara primestar max were used to amplify a 2 kb fragment from human genomic dna. High fidelity thermostable dna polymerase is a key requirement for all kinds of pcr applications. Datasheet for phusion highfidelity dna polymerase neb. Since pyrococcus furiosus is an extreme thermophile, its polymerase is active at. 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. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. Amplification of a short dna stretch by repeated cycles of in vitro dna polymerization science, vol 239, issue 4839, 487491. Thermostable phusion dna polymerase is isolated and purified from an li strain carrying a plasmid with the. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and phusion dna polymerase for sitedirected mutagenesis. The error rate of phusion hot start dna polymerase is equal to phusion dna polymerase, 4. In this study, we validated the pear technique for the preparation of 2. Next, aliquots of the reactions were treated with dpni and the dpnitreated and untreated reactions were transformed into the bl21 strain. Kapa hifi excels, phusion works sortof, q5 and platinum.
Guaninerich sequences inhibit proofreading dna polymerases. Phusion dna polymerase possesses the following activities. Thermo scientific phusion highfidelity dna polymerase 2. Phusion highfidelity dna polymerase, new england biolabs. We have found the enzyme to be highly robust and very accurate in our experiments.
We generally recommend using phusion dna polymerase at a concentration of 20 unitsml 1. Three types of rna polymerase in eukaryotic nuclei type location rna synthesized effect of. Amplification of a 500bp fragment using pfuultra highfidelity dna polymerase results in errors in less than 0. Pfu dna polymerase is an enzyme found in the hyperthermophilic archaeon pyrococcus furiosus, where it functions to copy the organisms dna during cell division. This protocol is for expressing and purifying the pfusso7d polymerase from e. The optimal amount of enzyme depends on the amount of template and the length of the pcr product. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear dna molecules with homologous ends. However, due to the novel nature of phusion hot start dna polymerase, optimal reaction conditions may differ from standard enzyme protocols. 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. The screen confirmed that very few templatecontaining colonies were. A method for copying a dna template, as well as a kit for performing the same, are also described. L reaction depending on the amplicon length and difficulty. Overlap extension pcr, phusion polymerase or taq polymerase. Phusion cannot incorporate dutp and is not recommended for use with uracilcontaining primers or template.
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