Phusion highfidelity dna polymerase thermo fisher scientific. It catalyzes the polymerization of nucleotides into duplex dna in the 5. Phusion sitedirected mutagenesis kit user guide pub. Due to the high salt concentration in the reaction buffer, phusion dna. It generates blunt ends in the amplification products. L phusion highfidelity dna polymerase, 2x phusion hf buffer in f531 or 2x phusion gc buffer in f532, and 400. These tubes hold a maximum of 50 l of reaction mix. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Phusion highfidelity dna polymerase high performance for. The tm of my primer pair using phusion hifi polymerase is 66 degrees c, as calculated by the neb tm calculator. Datasheet for phusion hot start flex dna polymerase. Overlap extension pcr, phusion polymerase or taq polymerase. Phusion hot start flex dna polymerase is unlike other enzymes and care must be taken when designing cycling protocols. Pcr protocol for phusion highfidelity dna polymerase m0530 overview the following guidelines are provided to ensure successful pcr using phusion dna polymerase.
Herculase ii fusion dna polymerase technical services us. If reactions are set up according to recommendations listed, the final concentration of phusion dna polymerase in the reaction is 1 unit50 l or 0. Primers should be at least 27 preferably 30 bases long with t m. The polymerase chain reaction pcr is the cardinal laboratory technology of molecular biology. It can also be used as a quikchangetype mutagenesis protocol. 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. First, the doublestranded dna template is denatured at a high temperature. 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. Thermostable phusion dna polymerase is isolated and purified from an e. Protocols optimized for phusion dna polymerase can directly be applied to phusion hot start dna polymerase reactions. Pcr reaction using phusion hot start ii dna polymerase f549. Thermo scientific phusion highfidelity pcr master mix product.
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. Herculase ii fusion dna polymerase technical services us and. A variant of this protein with an additional 65 amino acid changes is sold as phusion polymerase by new england biolabs. Using the modified cycling protocol described here, herculase ii fusion dna polymerase also provides.
Phusion hot start dna polymerase tends to work better at elevated denaturation and. New insights into the quikchangetm process guide the use of. This protocols is for pcr using q5 highfidelity dna polymerase m0491. 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. Pfuultra ii fusion hs dna polymerase technical services. The polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification. Component amount storage phusion hot start ii dna polymerase, 2 u. Protocol for a routine pcr with phusion highfidelity pcr. 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. Source organism phusion is extracted from extremophilic archaea.
Arguably one of the most powerful laboratory techniques ever discovered, pcr combines the unique attributes of being very sensitive and specific with a great degree of flexibility. 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. Moreover, the special enzyme formulation and optimized buffer system ensure robust performance when amplifying difficult and gcrich targets. 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. Prepare a master mix for the appropriate number of samples to be amplified. If tacloning is preferred, then dna should be purified prior to aaddition, as phusion dna polymerase will degrade any overhangs generated. Pfu dna polymerase possesses 3 to 5 exonuc lease proofreading activity that enables the polymerase to correct nucleotide misincorporation errors. 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. Otherwise, you will have problems with proper annealing. Polymerase does not require any separate activation step in the.
Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. Thermo scientific phusion highfidelity pcr master mix. Herculase ii fusion dna polymerase provides accuracy comparable to pfu dna polymerase. Taq polymerase, being thermostable, proved ideal for pcr. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification. 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. This is the pcr protocol for phusion highfidelity dna polymerase m0530. This pfu variant has the sso7d processivityenhancing domain attached that increases its speed and processivity. Thermo scientific phusion hot start ii highfidelity dna. This page presents all the information in polbase for phusion. Phusion highfidelity dna polymerase produce blunt end dna products.
Guidelines for using phusion dna polymerase ng to ensure homogeneity and improve recovery. One primer is 20 nt long and the other is 22 nt long with very similar gc content. Q5 highfidelity dna polymerase q5 q5 highfidelity dna polymerase neb k kod dna polymerase emd p phusion highfidelity dna polymerase neb pu pfuultra highfidelity dna polymerase agilent 4,960 bp 370 bp 410 bp 630 bp 520 bp m m m m 40% gc q5 p k pu 60% gc q5 p k pu 67% gc 72% gc 78% gc the standalone enzyme comes. Thermo scientific phusion hot start ii dna polymerase 2 ul. 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. If available, a representative structure is displayed at the. Dna polymerase tends to work better at elevated denaturation and annealing temperatures due. This protocol is for expressing and purifying the pfusso7d polymerase from e. This method has been tested with plasmids generally used in the lab i. Phusion dna polymerases produce blunt end dna products. In case of pcrs using the phusion dna polymerase, we employed the pcr protocol for whole blood. The pcr products generated using phusion dna polymerase have blunt ends.
Phusion highfidelity dna polymerase, new england biolabs. Please note that protocols with phusion dna polymerase may differ from protocols with other standard polymerases. Phusion hot start dna polymerase possesses the following activities. The dna polymerase should be pipetted carefully and gently as the high glycerol content 50 % in the storage buffer may otherwise lead to pipetting errors. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols.
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 using phusion hot start ii dna polymerase, it is not necessary to perform the pcr setup on ice. The five quality features of q5 high fidelity dna polymerase 1. At the top of the page youll find a map of all positions of known mutants. Pcr protocol for phusion highfidelity dna polymerase. Thermo scientific phusion highfidelity dna polymerases offer extreme. The concentration of phusion dna polymerase in the phusion pcr master mix has been optimized for best results under a wide range of conditions. Polymerase pcr protocol for phusion highfidelity dna.
Polymerase last, in order to prevent any primer degradation caused by the 3. Phusion green hot start ii highfidelity dna polymerase. However, due to the novel nature of phusion hot start dna polymerase, optimal reaction conditions may differ from standard enzyme protocols. Thermo scientific phusion rtpcr kit product information. Phusion dna polymerases thermo fisher scientific us. Protocol for a routine pcr with phusion highfidelity pcr kit introduction. Thermo scientific phusion hot start ii dna polymerase 2 u. Phusion highfidelity pcr master mix with hf buffer neb. 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. Thermo scientific phusion hot start ii highfidelity pcr master mix. Difference between phusion and taq polymerase compare.
Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol. Phusion highfidelity dna polymerase new england biolabs. Except in the case of negative controls, purified p. Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. The phusion dna polymerase has the ability to stabilize primertemplate hybridization. Scientists realized that thermostable heatstable dna polymerases would be needed for pcr to work efficiently. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. Pfu dna polymerase is a thermostable enzyme that replicates dna at 75c. The polymerase chain reaction pcr was developed by chemist kary mullis in the 1980s, as a means to make many copies of dna fragments. As such, conditions recommended below should be used for. Phusion highfidelity dna polymerase high performance for all. Pcr reaction using phusion hot start ii dna polymerase f.
Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Thermo scientific phusion highfidelity dna polymerase. Text links to mutants and digestion products are listed in the mutants section. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. Phusion dna polymerase in phusion hf buffer is determined to be 4. Pfu dna polymerase is an enzyme found in the hyperthermophilic archaeon pyrococcus furiosus, where it functions to copy the organisms dna during cell division. New phusion green hot start ii highfidelity dna polymerase is a combination of phusion hot start ii dna polymerase and 5x phusion green buffers. 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. Onetaq dna polymerase is an optimized blend of taq and deep vent dna polymerases for use with routine and difficult pcr experiments. The onetaq reaction buffers and high gc enhancer have been formulated for robust yields with minimal. Phusion dna polymerase possesses the following activities.
New insights into the quikchangetm process guide the use. Please read the quick guide to modify your protocol for optimal results. Rapid protocol times can be achieved with phusion flash highfidelity pcr master mix, a product developed especially for fast pcr. Highest fidelity dna amplification available at 280x higher than taq, q5 offers unparalleled fidelity for your most important samples, but with a protocol and pricepoint that makes it accessible. This simplifies the cdna synthesis protocol as it renders a separate rnase treatment step unnecessary. First, the doublestranded dna template is denatured at a high. 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. Protocol for phusion highfidelity pcr master mix with gc. This high processivity allows extremely short extension times and consequently reduced protocol times.
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. Pcr amplifies specific dna sequences exponentially by using multiple cycles of a threestep process. 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. Oct 19, 2012 in case of pcrs using the phusion dna polymerase, we employed the pcr protocol for whole blood. Control template and primers are provided for 20 control reactions. Difference between phusion and taq polymerase compare the.
1416 1536 481 1089 54 1622 1633 653 879 943 900 57 20 662 1649 556 1314 228 201 114 1581 482 1255 917 1630 1266 410 514 511 133 1445 303 569 983 848 116 1254 582