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 dna polymerase should be pipetted carefully and gently as the high glycerol content 50 % in the storage buffer may otherwise lead to pipetting errors. Component amount storage phusion hot start ii dna polymerase, 2 u. Thermostable phusion dna polymerase is isolated and purified from an e. 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. 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. Polymerase does not require any separate activation step in the. Oct 19, 2012 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. Thermo scientific phusion rtpcr kit product information. First, the doublestranded dna template is denatured at a high temperature. Phusion highfidelity dna polymerase high performance for. Pcr reaction using phusion hot start ii dna polymerase f549. Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. Phusion highfidelity dna polymerase, new england biolabs. 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. It can also be used as a quikchangetype mutagenesis protocol. Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol. Pfu dna polymerase is a thermostable enzyme that replicates dna at 75c. However, due to the novel nature of phusion hot start dna polymerase, optimal reaction conditions may differ from standard enzyme protocols.
It catalyzes the polymerization of nucleotides into duplex dna in the 5. As a result of this unique functional pairing, phusion dna polymerases are capable of generating pcr products with very high accuracy and speed. These tubes hold a maximum of 50 l of reaction mix. Thermo scientific phusion hot start ii highfidelity dna.
Herculase ii fusion dna polymerase provides accuracy comparable to pfu dna polymerase. Pcr protocol for phusion highfidelity dna polymerase m0530 overview the following guidelines are provided to ensure successful pcr using phusion dna polymerase. This simplifies the cdna synthesis protocol as it renders a separate rnase treatment step unnecessary. 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. One primer is 20 nt long and the other is 22 nt long with very similar gc content. Source organism phusion is extracted from extremophilic archaea. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Text links to mutants and digestion products are listed in the mutants section. L phusion highfidelity dna polymerase, 2x phusion hf buffer in f531 or 2x phusion gc buffer in f532, and 400.
Herculase ii fusion dna polymerase technical services us and. For the subsequent amplification of the cdna template. Phusion dna polymerase in phusion hf buffer is determined to be 4. Otherwise, you will have problems with proper annealing. Please note that protocols with phusion dna polymerase may differ from protocols with other standard polymerases. Guidelines for using phusion dna polymerase ng to ensure homogeneity and improve recovery. 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. Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. 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. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. Datasheet for phusion highfidelity dna polymerase neb.
Moreover, the special enzyme formulation and optimized buffer system ensure robust performance when amplifying difficult and gcrich targets. 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. The phusion dna polymerase has the ability to stabilize primertemplate hybridization. This method has been tested with plasmids generally used in the lab i. Except in the case of negative controls, purified p. This page presents all the information in polbase for phusion. Phusion dna polymerases produce blunt end dna products. At the top of the page youll find a map of all positions of known mutants. If tacloning is preferred, then dna should be purified prior to aaddition, as phusion dna polymerase will degrade any overhangs generated. This pfu variant has the sso7d processivityenhancing domain attached that increases its speed and processivity. 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.
Primers should be at least 27 preferably 30 bases long with t m. Phusion dna polymerase may be diluted in 1x hf or gc buffer just prior to use in order to reduce pipetting errors. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. 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 highfidelity dna polymerase produce blunt end dna products. Protocols optimized for phusion dna polymerase can directly be applied to phusion hot start dna polymerase reactions. 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. Thermo scientific phusion hot start ii dna polymerase 2 ul. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification. As such, conditions recommended below should be used for. Protocol for phusion highfidelity pcr master mix with gc. Phusion highfidelity dna polymerase new england biolabs. 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. Datasheet for phusion hot start flex dna polymerase.
Phusion sitedirected mutagenesis kit user guide pub. Pfu dna polymerase is an enzyme found in the hyperthermophilic archaeon pyrococcus furiosus, where it functions to copy the organisms dna during cell division. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. 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. When using phusion hot start ii dna polymerase, it is not necessary to perform the pcr setup on ice. 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. Thermo scientific phusion hot start ii highfidelity pcr master mix. Taq polymerase, being thermostable, proved ideal for pcr. If available, a representative structure is displayed at the. Phusion highfidelity pcr master mix with hf buffer neb. Phusion dna polymerase possesses the following activities. Difference between phusion and taq polymerase compare.
This protocols is for pcr using q5 highfidelity dna polymerase m0491. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. Pcr protocol for phusion highfidelity dna polymerase. The polymerase chain reaction pcr was developed by chemist kary mullis in the 1980s, as a means to make many copies of dna fragments. Thermo scientific phusion highfidelity pcr master mix.
A variant of this protein with an additional 65 amino acid changes is sold as phusion polymerase by new england biolabs. 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. This high processivity allows extremely short extension times and consequently reduced protocol times. Phusion green hot start ii highfidelity dna polymerase. Protocol for a routine pcr with phusion highfidelity pcr. Thermo scientific phusion highfidelity pcr master mix product. 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. Phusion dna polymerases thermo fisher scientific us. New insights into the quikchangetm process guide the use. Amplification of templates with high gc content, high secondary structure, low template concentrations or long amplicons may require further. Rapid protocol times can be achieved with phusion flash highfidelity pcr master mix, a product developed especially for fast pcr.
Phusion hot start dna polymerase possesses the following activities. Thermo scientific phusion hot start ii dna polymerase 2 u. It generates blunt ends in the amplification products. Polymerase last, in order to prevent any primer degradation caused by the 3. In case of pcrs using the phusion dna polymerase, we employed the pcr protocol for whole blood. The tm of my primer pair using phusion hifi polymerase is 66 degrees c, as calculated by the neb tm calculator. This protocol is for expressing and purifying the pfusso7d polymerase from e. Using the modified cycling protocol described here, herculase ii fusion dna polymerase also provides.
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. Dna polymerase tends to work better at elevated denaturation and annealing temperatures due. 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. Phusion highfidelity dna polymerase thermo fisher scientific. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Overlap extension pcr, phusion polymerase or taq polymerase. Onetaq dna polymerase is an optimized blend of taq and deep vent dna polymerases for use with routine and difficult pcr experiments.
Thermo scientific phusion highfidelity dna polymerase. The concentration of phusion dna polymerase in the phusion pcr master mix has been optimized for best results under a wide range of conditions. Thermo scientific phusion highfidelity dna polymerases offer extreme. Herculase ii fusion dna polymerase technical services us. Phusion highfidelity dna polymerase high performance for all. The pcr products generated using phusion dna polymerase have blunt ends. 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 polymerase chain reaction pcr is the cardinal laboratory technology of molecular biology. Phusion hot start flex dna polymerase is unlike other enzymes and care must be taken when designing cycling protocols. Pcr protocol for taq dna polymerase with standard taq buffer m0273. All that is required is the addition of template, primers and water. 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. Please read the quick guide to modify your protocol for optimal results.
The five quality features of q5 high fidelity dna polymerase 1. This is the pcr protocol for phusion highfidelity dna polymerase m0530. The polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification. 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. Pcr amplifies specific dna sequences exponentially by using multiple cycles of a threestep process. Polymerase pcr protocol for phusion highfidelity dna. Pcr reaction using phusion hot start ii dna polymerase f. Due to the high salt concentration in the reaction buffer, phusion dna. First, the doublestranded dna template is denatured at a high. Thermostable phusion dna polymerase is isolated and purified from an li strain carrying a plasmid with the.
Difference between phusion and taq polymerase compare the. Pfu dna polymerase possesses 3 to 5 exonuc lease proofreading activity that enables the polymerase to correct nucleotide misincorporation errors. Control template and primers are provided for 20 control reactions. Phusion hot start dna polymerase tends to work better at elevated denaturation and. 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.
1038 1207 716 979 467 588 141 182 192 469 616 921 1038 779 240 197 375 341 717 1232 293 233 1539 328 986 1168 1161 21 1462 201 1212 687 16 839 167 776 478 457 389 1293