Use the neb tm calculator to estimate an appropriate annealing temperature when using neb pcr products. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. The powerplex fusion system and all system components are manufactured in accordance with iso 18385. Phusion u hot start pcr master mix is convenient 2x mix designed to minimize the number of pipetting steps. It generates blunt ends in the amplification products. We generally recommend using phusion dna polymerase at a concentration of 20 unitsml 1. Herculase ii fusion dna polymerase technical services us. Phusion u hot start pcr master mix f533s and f533l is convenient 2x mix designed to minimize the number of pipetting steps. Fusion dna polymerase, highyield, high fidelity agilent. 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.
With this kit, the entire plasmid is amplified using phosphorylated primers that introduce the desired changes. Phusion highfidelity dna polymerase new england biolabs. 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. All necessary materials are provided to amplify str regions of human. Cdna synthesis, in vitro transcription and labeling were performed as instructed by the manufacturers user manual clontech. So i did the reaction with phusion at 70 degrees, and then at 63 when the first one didnt work. Thermo scientific phusion u dna polymerase is a novel engineered high fidelity enzyme developed using fusion technology. Phusion hot start flex dna polymerase new england biolabs. One primer is 20 nt long and the other is 22 nt long with very similar gc content. Phusion dna polymerase 2 ul 5x phusion hf buffer 5x phusion gc buffer dmso 50 mm mgcl 2 solution store at 5 to 30c. Phusion sitedirected mutagenesis kit catalog number f541 pub. Phusion dna polymerase in phusion hf buffer is determined to be 4. Difference between phusion and taq polymerase compare.
The following guidelines are provided to ensure successful pcr using phusion dna polymerase. Phusion highfidelity dna polymerase high performance for all. Herculase ii helps overcome pcr challenges and achieves robust yields by successfully amplifying a range of complex targets, including low abundance and high gc targets. This item has been discontinued and is no longer available. The formulation is based on takaras unique, highfidelity primestar hs dna polymerase combined with an elongation factor to provide efficient priming and extension, which greatly reduces the time required for annealing and extension steps. According to the neb gibson planner, after inputting that i will be using phusion, they told me to use 72 degrees as my tm. Dna polymerases are widely used enzymes in molecular biology techniques and are also naturally present in all organisms that undergo dna. The phusion dna polymerase ensures high fidelity for the exponential amplification. Thermo scientific phusion hot start ii dna polymerase 2 u. Phusion hot start dna polymerase guidelines for pcr reactions.
I have recently tried using circular polymerase extension cloning to clone a 200 bp insert into a 6. New insights into the quikchangetm process guide the use. Thermo scientific phusion hot start ii dna polymerase 2 ul. All necessary materials are provided to amplify str regions of human genomic dna, including a hotstart thermostable dna polymerase, which is a component of the powerplex fusion 5x master mix. Herculase ii fusion dna polymerase technical services us and. At the top of the page youll find a map of all positions of known mutants. Affibody inhibits the dna polymerase activity at room temperature and thus prevents the amplification of nonspecific products. 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. 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 polymerase was developed by finnzymes oy, now a part of thermo fisher scientific. Primestar max dna polymerasefast and highfidelity pcr. Therefore, hf buffer should be used as the default buffer for. Phusion dna polymerase was developed by finnzymes oy, now a part of thermo. Phusion u hot start dna polymerase is also available in the master mix and green formats.
Pfu dna polymerase is a thermostable enzyme that replicates dna at 75c. Pcr reaction setup for phusion thermo fisher scientific us. Difference between phusion and taq polymerase compare the. Control template and primers are provided for 20 control reactions.
Its excellent performance is achieved by superior proofreading ability due to a robust 3 5 exonuclease activity. More details can be found in the technical manuals for phusion dna polymerases. It combines the phusion dna polymerase and a reversibly bound, specific. Enter your username and we will send you a link to reset your password if an associated user is. Phusion u green format f556s and f556l is a combination of phusion u hot start dna polymerase and 5x phusion green buffers. The phusion highfidelity pcr kit contains a sufficient supply of phusion highfidelity dna polymerase, phusion hf and gc buffers, deoxynucleotides, magnesium chloride, dmso, and dna size standard to perform 50 reactions small or 200 reactions large.
Phusion hot start ii high fidelity dna polymerase is the most accurate hot start dna polymerase on the market. 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 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. 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. Component amount storage phusion hot start ii dna polymerase, 2 u. 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. Phusion highfidelity dna polymerase, new england biolabs. Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol. Powerplexr fusion system for use on the applied biosystems. In this video we will show you how to set up a pcr reaction usingthermo scientific phusion highfidelity dna polymerase.
The tm of my primer pair using phusion hifi polymerase is 66 degrees c, as calculated by the neb tm calculator. Herculase ii fusion dna polymerase provides accuracy comparable to pfu dna polymerase. Moreover, the special enzyme formulation and optimized buffer system ensure robust. Phusion dna polymerase is supplied with standard 5x. Only template and primers need to be added by the user. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. 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. New insights into the quikchangetm process guide the use of. Moreover, the special enzyme formulation and optimized buffer system ensure robust performance when amplifying difficult and gcrich targets. The phusion u hot start dna polymerase is an enzyme that is combined with a reversibly bound, specific affibody protein. 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. Pfuturbo dna polymerase replicates both plasmid strands with high.
How to set up a pcr reaction with phusion dna polymerases. I was wondering if anyone has ever seen large deletions 4050 bp occur when using phusion polymerase to clone a gene. Advanced biochemicals red hot dna polymerasea new rival for taq polymerase with convenience features. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. A 2x supermix is now available containing either hf buffer or gc buffer, dntps, and phusion polymerase. The tms should be calculated with the nearestneighbor method because results from primer tm calculations can vary significantly depending on the method. Phusion hot start dna polymerase guidelines for pcr. Phusion highfidelity pcr master mix is a convenient 2x mix containing phusion dna polymerase, nucleotides and optimized reaction buffer including mgcl 2. The purchase price of this product includes a limited, nontransferable license under u. Phusion sitedirected mutagenesis kit user guide pub. Phusion hot start dna polymerase has the ability to stabilize primertemplate hybridization. These results suggest that of the six commerciallyavailable dna polymerases we tested, kod. The formulation is based on takaras unique, highfidelity primestar hs dna. I have recently tried using circular polymerase extension cloning.
Manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity. Can i use phusion polymerase in quikchange protocol. Because my primers are so long, they have relatively high tms. Binding of the affibody protein inhibits dna polymerase and 35. Read the safety data sheets sdss and follow the handling instructions. According to the neb gibson planner, after inputting that i will be using phusion, they told. Phusion dna polymerases thermo fisher scientific us. Research multiple reassortment between pandemic h1n1 2009 manual on animal in. It catalyzes the polymerization of nucleotides into duplex dna in the 5. Binding of the affibody protein inhibits dna polymerase and 35 exonuclease activities at ambient temperatures, which allows you easy pipetting. View the product for possible alternatives or contact our product support team on 01509 555888 for assist. This page presents all the information in polbase for phusion. The recommendation for final primer concentration is 0.
Phusion hot start ii highfidelity enzyme is inactive at room temperature, due to the binding of a specific affibody ligand, which allows you to easily avoid the production of nonspecific. Primestar hs dna polymerase is a novel highfidelity dna polymerase that allows highefficiency amplification of large dna products up to 8. These results suggest that of the six commerciallyavailable dna polymerases we tested, kod fx dna polymerase is the most resistant to blood component inhibitors. Primestar max dna polymerase has the highest fidelity and fastest extension rate of any takara bio enzyme. It combines the phusion dna polymerase and a reversibly bound, specific affibody ligand. View the product for possible alternatives or contact.
Thermo scientific phusion sitedirected mutagenesis kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions in any type of plasmid dna. The phusion highfidelity pcr kit contains a sufficient supply of phusion highfidelity dna polymerase, phusion hf and gc buffers, deoxynucleotides, magnesium chloride, dmso, and. The quikchange sitedirected mutagenesis kit is used to make point mutations, switch amino acids, and delete or insert single or multiple amino acids. Thermo scientific ep0502 pfu dna polymerase recombinant 500u. 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. Other phusion hot start flex dna polymerase product introduction for high speed and high performance pcr. The quikchange sitedirected mutagenesis method is performed using pfuturbo dna polymerase and a temperature cycler. Thermo scientific phusion u hot start dna polymerase. Phusion highfidelity dna polymerase high performance for. The gc buffer can improve the performance of phusion dna polymerase on some difficult or long templates. Using the modified cycling protocol described here, herculase ii fusion dna polymerase also provides. 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 m0530 new england biolabs. One primer is 20 nt long and the other is 22 nt long with very similar gc.
142 13 1085 1495 1541 139 642 1295 240 731 1161 571 1407 395 399 1219 820 511 479 1154 1344 218 1033 784 6 767 47 1053 89 384 1141 377 1024