Phusion dna polymerase in phusion hf buffer is determined to be 4. Source organism phusion is extracted from extremophilic archaea. 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. 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 sitedirected mutagenesis kit user guide pub. All that is required is the addition of template, primers and water. Control template and primers are provided for 20 control reactions. However, due to the novel nature of phusion hot start dna polymerase, optimal reaction conditions may differ from standard enzyme protocols. Otherwise, you will have problems with proper annealing.
This simplifies the cdna synthesis protocol as it renders a separate rnase treatment step unnecessary. Phusion highfidelity dna polymerase, new england biolabs. Polymerase last, in order to prevent any primer degradation caused by the 3. This high processivity allows extremely short extension times and consequently reduced protocol times. This protocols is for pcr using q5 highfidelity dna polymerase m0491. Phusion dna polymerases incorporate more nucleotides per binding event as compared to other polymerases. Scientists realized that thermostable heatstable dna polymerases would be needed for pcr to work efficiently.
First, the doublestranded dna template is denatured at a high temperature. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Herculase ii fusion dna polymerase technical services us. Phusion highfidelity dna polymerase thermo fisher scientific.
Component amount storage phusion hot start ii dna polymerase, 2 u. 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 hot start dna polymerase tends to work better at elevated denaturation and. 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. Phusion hot start dna polymerase possesses the following activities. The onetaq reaction buffers and high gc enhancer have been formulated for robust yields with minimal. Protocol for a routine pcr with phusion highfidelity pcr kit introduction. Except in the case of negative controls, purified p. 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. Thermo scientific phusion highfidelity dna polymerase. Phusion highfidelity pcr master mix with hf buffer neb. Herculase ii fusion dna polymerase provides accuracy comparable to pfu dna polymerase. It generates blunt ends in the amplification products.
This is the pcr protocol for phusion highfidelity dna polymerase m0530. Protocol for a routine pcr with phusion highfidelity pcr. Protocols optimized for phusion dna polymerase can directly be applied to phusion hot start dna polymerase reactions. The tm of my primer pair using phusion hifi polymerase is 66 degrees c, as calculated by the neb tm calculator. The five quality features of q5 high fidelity dna polymerase 1. Rapid protocol times can be achieved with phusion flash highfidelity pcr master mix, a product developed especially for fast pcr. If available, a representative structure is displayed at the. One primer is 20 nt long and the other is 22 nt long with very similar gc content. This method has been tested with plasmids generally used in the lab i. Difference between phusion and taq polymerase compare the. New insights into the quikchangetm process guide the use.
Thermo scientific phusion hot start ii dna polymerase 2 u. Thermo scientific phusion hot start ii highfidelity pcr master mix. Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. 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. First, the doublestranded dna template is denatured at a high. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. If tacloning is preferred, then dna should be purified prior to aaddition, as phusion dna polymerase will degrade any overhangs generated. Pcr reaction using phusion hot start ii dna polymerase f549. Phusion highfidelity dna polymerase high performance for. Phusion dna polymerases thermo fisher scientific us. The pcr products generated using phusion dna polymerase have blunt ends.
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 pfu variant has the sso7d processivityenhancing domain attached that increases its speed and processivity. This page presents all the information in polbase for phusion. Polymerase does not require any separate activation step in the. Thermo scientific phusion highfidelity dna polymerases offer extreme. Phusion green hot start ii highfidelity dna polymerase. L phusion highfidelity dna polymerase, 2x phusion hf buffer in f531 or 2x phusion gc buffer in f532, and 400. Datasheet for phusion hot start flex dna polymerase. Guidelines for using phusion dna polymerase ng to ensure homogeneity and improve recovery. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification.
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. As such, conditions recommended below should be used for. Phusion dna polymerases produce blunt end dna products. 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. 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. In case of pcrs using the phusion dna polymerase, we employed the pcr protocol for whole blood. Prepare a master mix for the appropriate number of samples to be amplified. New insights into the quikchangetm process guide the use of. Thermostable phusion dna polymerase is isolated and purified from an e. The polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification. Protocol for phusion highfidelity pcr master mix with gc. Primers should be at least 27 preferably 30 bases long with t m.
As a result of this unique functional pairing, phusion dna polymerases are capable of generating pcr products with very high accuracy and speed. Onetaq dna polymerase is an optimized blend of taq and deep vent dna polymerases for use with routine and difficult pcr experiments. 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. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. It can also be used as a quikchangetype mutagenesis protocol. Phusion highfidelity dna polymerase produce blunt end dna products. The phusion dna polymerase has the ability to stabilize primertemplate hybridization. Herculase ii fusion dna polymerase technical services us and.
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. Datasheet for phusion highfidelity dna polymerase neb. Overlap extension pcr, phusion polymerase or taq polymerase. Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol. 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. Please note that protocols with phusion dna polymerase may differ from protocols with other standard polymerases. These tubes hold a maximum of 50 l of reaction mix. Moreover, the special enzyme formulation and optimized buffer system ensure robust performance when amplifying difficult and gcrich targets. Thermo scientific phusion highfidelity pcr master mix product. Pcr protocol for phusion highfidelity dna polymerase. Pcr protocol for taq dna polymerase with standard taq buffer m0273. Please read the quick guide to modify your protocol for optimal results. Phusion dna polymerase may be diluted in 1x hf or gc buffer just prior to use in order to reduce pipetting errors.
Due to the high salt concentration in the reaction buffer, phusion dna. 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. This protocol is for expressing and purifying the pfusso7d polymerase from e. Polymerase pcr protocol for phusion highfidelity dna.
The polymerase chain reaction pcr is the cardinal laboratory technology of molecular biology. Phusion dna polymerase possesses the following activities. Pfu dna polymerase is a thermostable enzyme that replicates dna at 75c. New phusion green hot start ii highfidelity dna polymerase is a combination of phusion hot start ii dna polymerase and 5x phusion green buffers. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. 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. The concentration of phusion dna polymerase in the phusion pcr master mix has been optimized for best results under a wide range of conditions.
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. Thermostable phusion dna polymerase is isolated and purified from an li strain carrying a plasmid with the. Pcr reaction using phusion hot start ii dna polymerase f. Thermo scientific phusion hot start ii highfidelity dna. Pfu dna polymerase possesses 3 to 5 exonuc lease proofreading activity that enables the polymerase to correct nucleotide misincorporation errors. It catalyzes the polymerization of nucleotides into duplex dna in the 5. For the subsequent amplification of the cdna template. Phusion hot start flex dna polymerase is unlike other enzymes and care must be taken when designing cycling protocols.
Taq polymerase, being thermostable, proved ideal for pcr. 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. Amplification of templates with high gc content, high secondary structure, low template concentrations or long amplicons may require further. Im planning to use overlap extension pcr for dna insertion into a vector, as it is explained by briskin and matsmura in. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons.
Thermo scientific phusion hot start ii dna polymerase 2 ul. Difference between phusion and taq polymerase compare. Pcr amplifies specific dna sequences exponentially by using multiple cycles of a threestep process. Phusion highfidelity dna polymerase high performance for all. Pcr protocol for phusion highfidelity dna polymerase m0530 overview the following guidelines are provided to ensure successful pcr using phusion dna polymerase.
Pfuultra ii fusion hs dna polymerase technical services. 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. Text links to mutants and digestion products are listed in the mutants section. 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. A variant of this protein with an additional 65 amino acid changes is sold as phusion polymerase by new england biolabs. 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.
Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. Dna polymerase tends to work better at elevated denaturation and annealing temperatures due. 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. Using the modified cycling protocol described here, herculase ii fusion dna polymerase also provides. Thermo scientific phusion highfidelity pcr master mix. 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. Thermo scientific phusion rtpcr kit product information. When using phusion hot start ii dna polymerase, it is not necessary to perform the pcr setup on ice. The polymerase chain reaction pcr was developed by chemist kary mullis in the 1980s, as a means to make many copies of dna fragments. Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. At the top of the page youll find a map of all positions of known mutants. Oct 19, 2012 in case of pcrs using the phusion dna polymerase, we employed the pcr protocol for whole blood. Phusion highfidelity dna polymerase new england biolabs.