We have found the enzyme to be highly robust and very accurate in our experiments. Usually 1 unit of phusion hot start dna polymerase per 50 l reaction volume gives good results, but optimal amounts could range from 0. Amplification of a short dna stretch by repeated cycles of in vitro dna polymerization science, vol 239, issue 4839, 487491. Pdf the impact of dna polymerase and number of rounds of. 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. Phusion highfidelity pcr master mix is convenient 2x mix containing phusion dna polymerase, nucleotides and optimized reaction buffer including mgcl. Usually after dna purification, 260280 ratio will ranging between 1,82 pure dna but.
This is the pcr protocol for phusion highfidelity dna polymerase m0530. Choose phusion polymerase from thermo for highly accurate, fast and robust pcr. Guaninerich sequences inhibit proofreading dna polymerases. Additionally, the user only needs to add template and primers minimizing the number. Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol. In our lab, finnzymes phusion dna polymerase was bought for cloning lengthy and. Amplification of a 500bp fragment using pfuultra highfidelity dna polymerase. It generates blunt ends in the amplification products. Because my primers are so long, they have relatively high tms. Wang department of pathology stanford university school of medicine stanford, california 943055324 enzymatic properties and characteristics that. Phusion dna polymerase in a total reaction volume of 50 l. Phusion dna polymerase from new england biolabs new england biolabs is pleased to announce that it will continue to provide its customers with phusion dna polymerase and related products, which have been distributed for the last six years on behalf of finnzymes oy. Pcr protocol for phusion highfidelity dna polymerase.
Highthroughput mutagenesis using a twofragment pcr approach. Phusion dna polymerase may be diluted in 1x hf or gc buffer just prior to use in order to reduce pipetting errors. Jun 18, 2015 taq polymerase was named after the thermophilic bacterium thermus aquaticus from which it was isolated in 1965 by thomas d. Readytouse 2x master mix preserves the fidelity and the yield in the reaction when using extremely short pcr protocols. The polymerase chain reaction polymerase chain reaction mullis, k. Thermo scientific phusion highfidelity pcr kit life. One unit is defined as the amount of enzyme that will incorporate 10 nmol of dntp into acid insoluble material in 30 minutes at 74c. Usually 1 unit of phusion hot start ii dna polymerase per 50. The annealing rules are different from many common dna polymerases such as taq dna polymerases. Phusion and iproof dna polymerases and 20fold higher than taq dna polymerase. Thermo scientific phusion rtpcr kit is a complete kit designed for performing cdna. I was wondering if anyone has ever seen large deletions 4050 bp occur when using phusion polymerase to clone a gene.
Readytouse 2x master mix preserves the fidelity and the yield in the reaction when using extremely short. New insights into the quikchangetm process guide the use of. According to the neb gibson planner, after inputting that i will be using phusion, they told me to use 72 degrees as my tm. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. The following guidelines are provided to ensure successful pcr using phusion dna polymerase.
Difference between phusion and taq polymerase compare the. In the figure, the myfi kit could amplify the stra sequence, which the phusion kit could not. Resuspend each cell pellet with 3 ml of lysis buffer and combine tubes together, mix well using pipette. Both phusion hf buffer and phusion gc buffer provide 1. The enzyme is a taq dna polymerase supplied in a proprietary formulation containing 50% glycerol with buffers designed for enhanced amplification.
Phusion highfidelity dna polymerase is supplied with 5x phusion hf buffer, 5x phusion gc buffer, dmso and 50 mm mgcl2 solution. The phusion highfidelity dna polymerase possesses the following activities. Phusion highfidelity dna polymerase new england biolabs. Phusion dna polymerase is supplied with standard 5x phusion hf. The optimal amount of enzyme depends on the amount of template and the length of the pcr product. Dna polymerases are widely used enzymes in molecular biology techniques and are also naturally present in all organisms that undergo dna replication. Combine the following components in reaction tubes. Datasheet for phusion highfidelity dna polymerase neb. Phusion, q5 and cobuddy are proofreading polymerases fused. In our lab, finnzymes phusion dna polymerase was bought for cloning lengthy and difficult dna strands.
L reaction volume gives good results, but the optimal amount can range from 0. Please note that protocols with phusion dna polymerase may differ from protocols with other standard polymerases. Amplification of a 500bp fragment using pfuultra highfidelity dna polymerase results in errors in less than 0. Protocol for harvesting pfusso7d polymerase barrick lab. Phusion dna polymerase is a very high fidelity polymerase which is artificially constructed by dna binding domain to proofreading polymerase from pyrococcus furiosus. The impact of dna polymerase and number of rounds of amplification in pcr on 16s rrna gene sequence data article pdf available may 2019 with 61 reads how we measure reads. The polymerase blend is very powerful for pcr amplifying of long dna fragment. 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. The enzyme is a taq dna polymerase supplied in a proprietary. Thermo scientific phusion flash pcr master mix was developed to save valuable laboratory time.
Ultramer pcr reactions were performed in a 20l volume. Datasheet for phusion highfidelity dna polymerase m0530. Dna polymerase and 18fold lower than taq dna polymerase, making it the highest fidelity enzyme available see table i. Phusion did not amplify a plasmid with complementary primers but. Phusion highfidelity dna polymerase high performance for. Overlap extension pcr, phusion polymerase or taq polymerase. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification.
According to the new understanding, we developed a new protocol with phusion dna polymerase and. New insights into the quikchangetm process guide the use. Im doing dna extraction using chelex and before dna purification, it have 260280 ratio start from 1,11,4. The phusion high fidelity dna polymerase is also capable of amplifying long amplicons such as the 7. Therefore, the hf buffer should be used as the default buffer for highfidelity amplification. Specific synthesis of dna in vitro via a polymerase catalysed chain reaction. Im planning to use overlap extension pcr for dna insertion into a vector, as it is explained by briskin and matsmura in. Jan 23, 2006 high fidelity thermostable dna polymerase is a key requirement for all kinds of pcr applications. Phusion requires at least 15 seconds per kb during the elongation phases. 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. The quikchangetm manual recommends continuing with.
The enzyme, originally developed by finnzymes, is a fusion of. High fidelity thermostable dna polymerase is a key requirement for all kinds of pcr applications. Phusion dna polymerase gave strong specific bands even with the shortest extension time, completing the 3. 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. 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 5x green gotaq reaction buffer contains two dyes blue and. If available, a representative structure is displayed at the.
These results suggest that of the six commerciallyavailable dna polymerases we tested, kod. We generally recommend using phusion dna polymerase at a concentration of 20 unitsml 1. Thermo scientific phusion highfidelity pcr kit 500u. 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 polymerase pcr reactions pcr amplification of long sequences was performed using phusion hot start dna polymerase 2ul finnzymes. According to the neb gibson planner, after inputting that i will be using phusion, they told me to use. Phusion dna polymerase in phusion hf buffer is determined to be 4. This page presents all the information in polbase for phusion. Enzyme amounts also indicate that significantly less of the highly processive phusion dna polymerase is required to complete the task. L reaction depending on the amplicon length and difficulty. Three types of rna polymerase in eukaryotic nuclei type location rna synthesized effect of. Phusion highfidelity dna polymerase high performance for all.
Difference between phusion and taq polymerase compare. 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. Reactions can also be optimized using the provided dmso or mgcl 2 solutions. Phusion dna polymerases the best choice for highfidelity cloning. So i did the reaction with phusion at 70 degrees, and then at 63 when the first one didnt work. When cloning fragments amplified with phusion dna polymerases, blunt end. Taq polymerase was named after the thermophilic bacterium thermus aquaticus from which it was isolated in 1965 by thomas d. Phusion dna polymerase possesses the following activities. 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. 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 highfidelity dna polymerase phusion hot start ii highfidelity dna polymerase phusion flash highfidelity dna polymerase phusion u hot start dna polymerase phusion u multiplex pcr master mix characteristics blunt or 3a end blunt blunt blunt blunt blunt target length, genomicphage dna. Thermo scientific phusion green highfidelity dna polymerase. However, the optimal concentration of phusion dna polymerase may vary from 1040 unitsml 0. The myfi polymerase could amplify complicated short tandem repeat str or ssrmicrosatellite sequences better than the other kits.
Phusion highfidelity dna polymerase thermo fisher scientific. 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. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. At the top of the page youll find a map of all positions of known mutants. Phusion highfidelity dna polymerase, new england biolabs. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. Text links to mutants and digestion products are listed in the mutants section. However, gc buffer can improve the performance of phusion dna polymerase on some difficult or long templates, such as gcrich templates or those with. We commonly use the bioline myfi polymerase, and it works better than any other polymerase kit. 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.
I have recently tried using circular polymerase extension cloning to clone. In pcr amplification using phusion dna polymerase, how to. 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. 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.
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