The correct understanding is important to method improvements, guiding us to use partially overlapping primers and phusion dna polymerase for sitedirected mutagenesis. Phusion hot start ii highfidelity dna polymerase is capable of amplifying long amplicons such as the 7. Recently ive been trying to amplify 4kb using the phusion polymerase, but i have doubts with the annealing temperature, can somebody recomend an article talking about that. Datasheet for phusion highfidelity dna polymerase neb. Yongzhen xia, wenqiao chu, qingsheng qi, luying xun. New insights into the quikchangetm process guide the use. Reactions can also be optimized using the provided dmso or mgcl 2 solutions. Thermo scientific phusion hot start ii highfidelity dna. The linear dna molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in e.
What are the advantagesdisadvantages of phusion vs taq. The following link is to a pdf safety data sheet sds that applies to this product to help you use it safely. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. Examples of these include nebs phusion polymerase, geneons onefusion dna polymerase and agilents pfuultra ii fusion hs dna polymerase. For selecting a polymerase, most companies will provide y.
Thermostable phusion dna polymerase is isolated and purified from an li strain carrying a plasmid with the. Herculase ii fusion dna polymerase technical services us. Since pyrococcus furiosus is an extreme thermophile, its polymerase is active at. Phusion highfidelity dna polymerase new england biolabs. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Download a pdf containing pricing for our full product list.
If tacloning is preferred, then dna should be purified prior to aaddition, as phusion dna polymerase will degrade any overhangs generated. It generates blunt ends in the amplification products. The pcr products generated using phusion dna polymerase have blunt ends. Moreover, the special enzyme formulation and optimized buffer system ensure robust performance when amplifying difficult and gcrich targets. Dna polymerase, 5x phusion hf buffer, dntps, quick t4 dna ligase, 2x. The pcr reactions were also done with commercially available pfuturbo agilent, primestar takara, transtaq transgene, phusion fisherthermo scientific, pyrobest dna polymerase takara, kod fx dna polymerase toyobo life science and q5 dna polymerase new england lab in 20. 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 fidelity of a dna polymerase is the result of accurate. New insights into the quikchangetm process guide the use of phusion dna polymerase for sitedirected mutagenesis. L of the 2x master mix depending on the final reaction volume. Difference between phusion and taq polymerase compare.
Using a lacibased method modified from previous studies1, the error rate of. Dna used in thermo fisher scientific quality control assays. New insights into the quikchange tm process guide the use of phusion dna polymerase for sitedirected mutagenesis. Phusion the wolfson centre for applied structural biology. The phusion sitedirected mutagenesis kit includes phusion hot start. Thermo scientific phusion highfidelity dna polymerase. Phusion dna polymerase is a very high fidelity polymerase which is artificially constructed by dna binding domain to proofreading polymerase from pyrococcus furiosus. Pfubased dna polymerase with a high affinity doublestranded dna binding domain. Anneal at 3c above the lowest tm if the primers are longer than 20nt, else at the tm.
Taq dna polymerase discovery leads to the pcr invention. Typically the length of such primers is 20 nt or more. However, taq polymerase is still used as the standard polymerase enzyme in pcr. Phusion hot start dna polymerase tends to work better at elevated denaturation and. Phusion highfidelity dna polymerase high performance for.
Affibody inhibits the dna polymerase activity at room temperature and thus prevents the amplification of nonspecific products. This is the difference between phusion and taq polymerase. Phusion dna polymerase in phusion hf buffer is determined to be 4. It carries the weighty responsibility of duplicating our genetic information. Finnzymes phusion highfidelity dna polymerase offers extreme performance for all pcr applications. Using the modified cycling protocol described here, herculase ii fusion dna polymerase also provides. We enable science by offering product choice, services, process excellence and our people make it. If you have a ridiculously long sequence of up to 25 kb dont fret. Thermo scientific phusion rtpcr kit product information. Phusion dna polymerase may be diluted in 1x hf or gc buffer just prior to use in order to reduce pipetting errors.
In addition, phusion dna polymerases are tolerant of various inhibitors. Learn more about phusion highfidelity dna polymerase. Each time a cell divides, dna polymerase duplicates all of its dna, and the cell passes one copy to each daughter cell. Pmc images search for phusion highfidelity dna polymerase. Phusion green hot start ii highfidelity dna polymerase. The phusion high fidelity dna polymerase possesses the following activities. It combines the phusion dna polymerase and a reversibly bound, specific affibody ligand. Dna polymerase plays the central role in the processes of life. Due to the unique nature of phusion dna polymerases, please pay special attention to the guidelines listed below. The thermo scientific phusion highfidelity dna polymerase offers high performance for all major pcr applications. L total if you are using any of the phusion pcr master mix products, add 25 or 10. However, due to the novel nature of phusion hot start dna polymerase, optimal reaction conditions may differ from standard enzyme protocols. Control template and primers are provided for 20 control reactions. Pfuultra ii fusion hs dna polymerase technical services.
With phusion hot start dna polymerase, use primers with tm 60c or higher. 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. Pdf new insights into the quikchangetm process guide the. Protocols optimized for phusion dna polymerase can directly be applied to phusion hot start dna polymerase reactions. In addition to being faster than taq, phusion also makes fewer errorskb on average at least, compared to a standard taq polymerase there are various high fidelity variants out there. Highfidelity dna polymerases are important for applications in which the dna sequence needs to be correct after amplification. 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. The tm values should be calculated with the nearestneighbor method5, because results from primer tm calculations can vary signi. Phusion highfidelity dna polymerase, new england biolabs.
Enhanced processivity and the inclusion of our exclusive archaemaxx pcr. Phusion dna polymerase, 5x phusion hf buffer, 5x phusion gc buffer, dntp mix, mgcl2 solution, control lambda dna, 1. Herculase ii fusion dna polymerase provides accuracy comparable to pfu dna polymerase. Pcr protocol for phusion highfidelity dna polymerase. Phusion hot start ii high fidelity dna polymerase combines the phusion dna polymerase and a reversibly bound, specific affibody ligand. Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. Thermo scientific phusion highfidelity dna polymerase 2. Phusion has many advantages over taq that has made phusion a better option in producing high fidelity dna. Phusion dna polymerases thermo fisher scientific us. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. The result is an enzyme mix that amplifies genomic targets in excess of 20kb. Our lab dntp stocks contain 10 mm each of datp, dttp, dctp, and dgtp. Phusion highfidelity dna polymerase thermo fisher scientific. Manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr applications.
Thermo scientific phusion highfidelity pcr kit life. A 2x supermix is now available containing either hf buffer or gc buffer, dntps, and phusion polymerase. Thermo scientific phusion hot start ii dna polymerase 2 u. This is the pcr protocol for phusion highfidelity dna polymerase m0530. Make sure to keep the enzymes and dntp stocks on ice when taken outside the freezer. Phusion dna polymerase possesses the following activities.