I was able to easily pcr amplify 23kb inserts accurately. If the final volume is scaled down to 10 l, how many units of polymerase should be taken. Phusion dna polymerase may be diluted in 1x hf or gc buffer just prior to use in order to reduce pipetting errors. When using phusion polymerase, should i use an annealing. I have got wonderful results with 10 l volume using reagents from a different company. Pcr annealing temperatures and when using different dna polymerases and buffers. For high gc templates q5 dna polymerase with gc enhancer can be used for successful amplification. Phusion highfidelity dna polymerase is supplied with 5x phusion hf buffer, 5x phusion gc buffer, dmso and 50 mm mgcl2 solution. For optimal results start by accurately calculating your tm with our tm calculator. Jan 30, 2015 the linear dna molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in e. Performing calculations for large scale pcr reactions can be cumbersome and tedious.
L reaction volume gives good results, but the optimal amount can range from 0. At 5565c they anneal to the dna at 72 taq is most active and extends. Both phusion hf buffer and phusion gc buffer provide 1. Phusion flash pcr master mix was developed to save valuable laboratory time.
This tool calculates the tm of primers and estimates an appropriate. When using phusion polymerase, should i use an annealing temp. 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 phusion highfidelity dna polymerase sets a new standard for pcr performance. Phusion dna polymerases thermo fisher scientific us. Phusion dna polymerases from thermo fisher scientific description one of the highest referenced polymerase lines in thousands of publications, the thermo scientific phusion highfidelity polymerases are highly accurate and robust products that provide high yields with short reaction times and enhanced specificity. Pmc images search for phusion highfidelity dna polymerase figure 1 engineered proofreading dna polymerases have good performance.
Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification. A 3,874 bp target was pcr amplified with either taq thermopol buffer, q5 q5 reaction buffer with or without gc enhancer or phusion phusion hf buffer dna polymerase. Using phusion hot start ii highfidelity dna polymerase, amplification proceeds without the production of nonspecific products due to the combination of phusion dna polymerase and a reversibly bound, specific affibody ligand that inhibits dna polymerase activity at room temperature. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. Taq with standard taq buffer is available in economical extralarge pack sizes. However, taq polymerase is still used as the standard polymerase enzyme in pcr. Thermo scientific phusion sitedirected mutagenesis kit, point mutations, insertions and deletions can be introduced in any type of plasmid dna. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. Re 1990 optimization of the annealing temperature for dna amplification in vitro. When using phusion polymerase, should i use an annealing temp, which around the calculated tm of the primers pair. 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. Difference between phusion and taq polymerase compare the. Pcr using phusion high fidelity dna polymerase biocompare. Thermo scientific phusion u hot start dna polymerase is a modified phusion dna polymerase which efficiently amplifies uracilcontaining templates.
What are the advantagesdisadvantages of phusion vs taq. 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. L total if you are using any of the phusion pcr master mix products, add 25 or 10. Shop online for a wide selection of thermo scientific phusion green highfidelity dna polymerase 2 ul the most accurate thermostable dna polymerase available offering superior. The primer tm for phusion is calculated based on the breslauer method and is higher than the tm using taq or pfu polymerases. In this study, q5 was examined to determine its fidelity compared to taq dna polymerase using the two methods described below figure 2. Id like to clone a fragment amplified with phusion highfidelity dna polymerase. Phusion sitedirected mutagenesis kit user guide pub. To determine the optimal annealing temperatures for a given set of primers, use of the calculator. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna. Moreover, new england biolabs webpage has included a tm calculator to determine. Highfidelity dna polymerases are important for applications in which the dna sequence needs to be correct after amplification.
Thermo scientific phusion flash highfidelity pcr master. Which is the best online melting temperature tm calculator. 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. Phusion is a registered trademark and property of thermo. There are different published protocols to develop. To use the calculator select your dna polymerase, type in or paste your primer. In addition to high accuracy, pfuultra highfidelity dna polymerase provides robust amplification of long, complex genomic targets. Molecular cloning using polymerase chain reaction, an. The calculator also calculates the primer length, percentage of gc content, molecular weight, and extinction coefficient. Phusion highfidelity dna polymerase, new england biolabs. It generates blunt ends in the amplification products.
Use the neb tm calculator to estimate an appropriate annealing temperature. It is a proofreading dna polymerase that contains a unique. Why are there low molecular weight discrete bands on an agarose gel after a pcr using phusion highfidelity dna polymerase. Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. Phusion dna polymerases from thermo fisher scientific. Thermo scientific phusion green highfidelity dna polymerase. As a result of this unique functional pairing, phusion dna polymerases are capable of generating pcr products with very high accuracy and speed. As far as gc content the products were all only around 5560% gc so havent stressed the system too much. Taq dna polymerase is the industry standard for routine pcr.
How do you calculate the annealing temperature for pcr. Thermo scientific phusion highfidelity pcr master mix. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and phusion dna polymerase for sitedirected mutagenesis. A separate method is used for t m and annealing temperature calculation of reactions with taqbased dna polymerases. Pfu dna polymerase is a thermostable enzyme that replicates dna at 75c. Choose phusion polymerase from thermo for highly accurate, fast and robust pcr.
As a result of this unique functional pairing, phusion dna polymerases. In the quality control tests of phusion dna polymerase, 7. Instructions for tm calculation and a link to a calculator. To determine the optimal annealing temperatures for a given set of primers, use of the calculator m neb t is highly recommended. A 2x supermix is now available containing either hf buffer or gc buffer, dntps, and phusion polymerase. Hot start ii dna polymerase is equal to that of phusion dna polymerase 4.
This product is manufactured by new england biolabs, inc. Product information thermo scientific phusion site. To use the calculator select your dna polymerase, type in or paste your primer sequences, and provide your final primer concentration. The annealing temperature ta chosen for pcr relies directly on length and. Taq dna polymerase discovery leads to the pcr invention. Neb tm calculator is used to estimate an appropriate annealing temperature in neb pcr products.
The phusion dna polymerase has the ability to stabilize primertemplate hybridization. Anneal at 3c above the lowest tm if the primers are longer than 20nt, else at the tm. T m values, annealing temperature, and other data are automatically generated. New insights into the quikchange tm process guide the use of. For phusion dna polymerases, the salt correction of schildkraut 2 is used. This kit uses the highly processive thermo scientific phusion hot start ii highfidelity dna polymerase. Phusion highfidelity pcr master mix is convenient 2x mix containing phusion dna polymerase. A testing various dna polymerases for the amplification of a 1 kb fragment with 70% gccontent. Calculation of optimal annealing temperature fastpcr manual. This online tool will calculate the amounts of components needed to create your pcr master mix. The purchase price of this product includes a limited, non. Degenerated nucleotides are allowedbasestacking tm. T4 dna ligase gotaq g2 dna polymerase pgemt easy vector systems. The calculator calculates recommended t m melting temperature of primers and pcr annealing temperature based on the primer pair sequence, primer concentration, and dna polymerase used in pcr.
The master mix utilizes phusion flash ii dna polymerase, a proofreading dna polymerase modified from phusion hot start ii highfidelity dna polymerase. Difference between phusion and taq polymerase compare. Get a complimentary sample of thermo scientific phusion highfidelity dna polymerase when you fill out the form on this page. Calculation of taq polymerase pcr, rtpcr and realtime pcr. Determine your primers optimal annealing temperature. Consider only the gene specific bases for tm calculation and leave the 5 prime extensions. The unique structure and characteristics of phusion dna polymerase make it a superior choice for cloning. You should use neb tool for annealing temperature calculation. Phusion highfidelity dna polymerase high performance for all. The tm of my primer pair using phusion hifi polymerase is 66 degrees c, as calculated by the neb tm calculator.
Sep 20, 2017 phusion dna polymerase is a very high fidelity polymerase which is artificially constructed by dna binding domain to proofreading polymerase from pyrococcus furiosus. Thermo scientific phusion flash highfidelity pcr master mix. The software will process the line as having an invalid second primer and will not. Phusion dna polymerase was developed by finnzymes oy, now a part of thermo fisher scientific. This is the difference between phusion and taq polymerase. The structure of phusion highfidelity dna polymerase.
Application calculates the tm of primers and estimates an appropriate annealing temperature when using different dna polymerases. One of the methods most commonly used to determine the impact of mutations is the site. Phusion has many advantages over taq that has made phusion a better option in producing high fidelity dna. The enzyme, originally developed by finnzymes, is a fusion of a proofreading polymerase and a processivityenhancing domain, creating one of the most reliable and robust polymerases available. The t m calculator is not required for platinum ii taq dna polymerase, platinum superfi. The dna is extracted from bacterial colonies by boiling. Phusion dna polymerase possesses the following activities. Dec 05, 2014 in this video we will show you how to set up a pcr reaction usingthermo scientific phusion highfidelity dna polymerase. Fastpcr is a free software for microsoft windows and is based on a new. Typically the length of such primers is 20 nt or more. Titanium taq dna polymerase is suitable for use in all pcr applications and with a wide range of samples, including bacterial and plasmid dna, cdna, and complex genomic dna. Application calculates the tm of primers and estimates an appropriate annealing. Using phusion hot start ii highfidelity dna polymerase, amplification proceeds without the production of nonspecific products due to the combination of phusion dna polymerase and a reversibly bound, specific affibody ligand that inhibits dna polymerase.
Hi, ive used phusion, but i guess i havent tested it out to much. A single unit of phusion dna polymerase produced higher yields than 2. Due to their high accuracy, robustness, reliability and inhibitor tolerance, phusion highfidelity dna. Primer 650 bases basic tm degenerated nucleotides are allowed. 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. Use the neb tm calculator to estimate an appropriate annealing temperature when using neb pcr products. Phusion flash highfidelity pcr master mix is a 2x master mix enabling the use of highly short pcr protocols without compromising either the fidelity or the yield in the reaction. Phusion hot start flex dna polymerase is unlike other enzymes and care must be taken when designing cycling protocols. 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. It catalyzes the polymerization of nucleotides into duplex dna in the 5. Phusion highfidelity dna polymerase high performance for. Phusion highfidelity pcr master mix is a convenient. Request a sample of thermo scientific phusion dna polymerase.
Tm calculator oligo melting temperature promega corporation. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. Phusion dna polymerase is a chimeric pfu which has a dna binding protein at the nterminal portion of the gene. It should be noted that phusion has different temperature requirements than other dna polymerases. A testing various dna polymerases for the amplification of a. One primer is 20 nt long and the other is 22 nt long with very similar gc content. Product information thermo scientific phusion rtpcr kit. The optimal annealing temperature ta is the range of temperatures where. This is the pcr protocol for phusion highfidelity dna polymerase m0530. Ensure your success of scaled up reactions by using the pcr master mix calculator. Will phusion highfidelity dna polymerase incorporate dutps. 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.
This enzyme keeps significant activity after exposure to 99c or repeated exposure to 98c with more processivity and extention rate than pfu dna polymerase. The tm values should be calculated with the nearestneighbor method5, because results from primer tm. Since genomic data are widely available, many strategies have been implemented to reveal the function of specific nucleotides or amino acids in promoter regions or proteins, respectively. Phusion highfidelity dna polymerases product listing product overview 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. Phusion hot start ii dna polymerase has the ability to stabilize primertemplate hybridization. In this video we will show you how to set up a pcr reaction usingthermo. It can be used to minimize carryover contamination in conjunction with dutp and uracil dna glycosylase udg and for amplification of bisulfitetreated and damaged dna. Note that the optimal annealing temperature for phusion hot start ii dna polymerase may differ significantly.
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