These results suggest that of the six commerciallyavailable dna polymerases we tested, kod fx dna polymerase is the most resistant to blood component inhibitors. All that is required is the addition of template, primers and water. Phusion highfidelity dna polymerase high performance for. If ta cloning is required, it can be performed by adding a overhangs to the blunt pcr product with. Define if you would like to add some extra volume for pipetting.
Pcr from ffpe tissues without dna extraction using phusion. Live imaging of intra and extracellular ph in plants. Thermo scientific phusion u multiplex pcr master mix. Overlap extension pcr cloning, described here, is not the first form of pcrmediated cloning 810. Because my primers are so long, they have relatively high tms. Im planning to use overlap extension pcr for dna insertion into a vector, as it is explained by briskin and matsmura in. Phusion highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr applications.
As a result of this unique functional pairing, phusion dna polymerases are capable of generating pcr products with very high accuracy and speed. Phusion blood direct pcr kit thermo fisher scientific. This was demonstrated with a standard pcr, with or without dttp replaced by dutp. Infusion advantage protocol overview the table below is a general outline of the protocol used. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Reactions can also be optimized using the provided dmso or mgcl 2 solutions. The modified phusion hot start ii highfidelity dna polymerase exhibits high resistance to pcr inhibitors found in blood an.
New insights into the quikchangetm process guide the use. Guidelines for pcr optimization with phusion highfidelity. The modified phusion hot start ii highfidelity dna polymerase exhibits high resistance to pcr inhibitors found in blood and retains polymerase activity even with samples containing 40% whole blood. Although some additional highmolecularweight bands over 2,000 base pairs were produced using kod fx dna polymerase, no primer dimers under 100 base pairs were detected. Joining pcr using q5 polymerase or pcr extender if phusion doesnt work. Usually after dna purification, 260280 ratio will ranging between 1,82 pure dna but. Amplification of templates with high gc content, high secondary structure, low template concentrations or long amplicons may require further optimization. Phusion hot start flex 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. Pcr reaction setup for phusion thermo fisher scientific us. Thermo scientific phusion blood direct pcr kit is developed for amplification of dna from whole blood. In general, add the water first and the enzyme last. Overlap extension pcr, phusion polymerase or taq polymerase. The phusion dna polymerase has the ability to stablize primertemplate hybridization.
It has a proof reading capability which removes any as added to the end. The polymerase is suitable also for amplification of long amplicons such as 7. For visual image presentation of ratio images, a pseudocolour look. Dont use phusion for any normal pcr, just because your pcr isnt working. Polymerase chain reaction, 122004 5 mgcl 2 the concentration of mgcl 2 influences the stringency of the interaction between the primers and the template dna. Pcr reaction setup with phusion dna polymerases fill in the volume of template dna you want to use, the number of pcr reactions you intend to run, and the individual reaction volume. Amplification of templates with high gc content, high secondary structure, low template concentrations or longer amplicons may require further optimization. A mutant pfu dna polymerase designed for advanced uracil. Colony pcr with phusion jul312008 hi everyone, i will appreciate any help. The result is an optimized protocol for the generation of p2a fusion genes that relies on the use of a high fidelity dna polymerase with high processivity. Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult amplicons.
Collect all liquid to the bottom of the tube by a quick spin if necessary. Phusion dna polymerases thermo fisher scientific us. It is technically difficult to amplify targets 5000 bp long. In the experiment, pfux7 was compared to the other highly processive phusion polymerase using standard phusion pcr conditions. It is a proprietary formulation that gives much better enzyme performance. Thermo scientific phusion rtpcr kit product information. The polymerase possesses a 3 5 exonuclease activity. Fill in the volume of template dna you want to use, the number of pcr reactions you intend to run, and the individual reaction volume. According to the neb gibson planner, after inputting that i will be using phusion, they told me to use 72 degrees as my tm. B gel electrophoresis of the pcr products produced by phusion.
Dna polymerase for exponential pcr amplification of dsdna plasmid to be mutated. The tms should be calculated with the nearest neighbor method as results from primer tm calculations can vary significantly depending on the method used. Its unique structure, a novel pyrococcus like enzyme fused with a processivityenhancing domain, increases fidelity and speed. 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. This dna polymerase exhibits exceptional accuracy in proofreading and makes the phusion blood direct pcr kit one of the best choices for demanding applications, such as cloning. Datasheet for phusion highfidelity dna polymerase neb. Phusion highfidelity pcr master mix is a convenient 2x mix containing phusion dna polymerase, nucleotides and optimized reaction buffer including mgcl 2. R is the ratio at a given ph, and r max and r min designate the maximum and minimum ratio obtainable at either high or low ph, respectively. Phusion the wolfson centre for applied structural biology. The polymerase chain reaction can be used to amplify both double and single stranded dna. Phusion flash pcr master mix is designed to give optimal results. The following guidelines are provided to ensure successful pcr using phusion dna polymerase.
Overlay the sample with mineral oil if using a pcr machine without a heated lid. Once the pcr is complete, run on a gel to confirm whether the genes have fused. Pfux7 accepts the replacement of dttp with dutp in pcr, and therefore is compatible with ungbased carry over contamination prevention. Im doing dna extraction using chelex and before dna purification, it have 260280 ratio start from 1,11,4. Phusion does not add as to the end of the amplicon. Optimization of overlap extension pcr for efficient transgene. This means if you want to then subclone you will have to add the as yourself. The reason is that these polymerases are fused to a processivityenhancing dsdnabinding domain that stabilizes the primer. The unique phusion highfidelity dna polymerase offers robust performance and can be used for all pcr applications. Kary mullis, for which he received the nobel prize in chemistry in 1993. Pcr reaction setup with phusion dna polymerases this calculator generates a pipetting table for setting up pcr reactions with thermo scientific phusion dna polymerases. Its unique structure, a novel pyrococcuslike enzyme fused with a processivityenhancing domain, increases fidelity and speed. 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. In the beginning i was trying to sequence the pcr fragment directly from phusion pcr.
Control template and primers are provided for 20 control reactions. L phusion highfidelity dna polymerase, 2x phusion hf buffer in f531 or 2x phusion gc buffer in f532, and 400. When cloning fragments amplified with phusion flash ii dna polymerase, blunt end cloning is recommended. Product information thermo scientific phusion fidelity pcr. Protocol for a routine pcr with phusion highfidelity pcr. Shop online for a wide selection of emd millipore novagen kod hot start dna polymerase for pcr amplification of long strand and gcrich dna templates. In pcr amplification using phusion dna polymerase, how to. Thermostable phusion dna polymerase is isolated and purified from an e. Pcr, the quick, easy method for generating unlimited copies of any fragment of dna, is one of those scientific developments that actually deserves timeworn superlatives like revolutionary and breakthrough. We enable science by offering product choice, services, process excellence and our people make it.
Windows can go online to look it up automatically, or you can manually select from a list of programs that are installed on your computer. Pcr protocol for phusion highfidelity dna polymerase. Pcr the polymerase chain reaction polymerase chain reaction mullis, k. It eliminates the need for a separate dna purification step prior to pcr. Slope describes the steepness of the curve, a larger value giving a shallower curve. Specific synthesis of dna in vitro via a polymerase catalysed chain reaction. Thermo scientific phusion highfidelity pcr master mix. Taq dna polymerase is relatively processive 60% that of phusion, but the overall. Phusion highfidelity dna polymerase thermo fisher scientific. Generally, pcr amplifies small dna targets 100 base pairs bp long. The combined use of restriction enzymes with pcr has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated dna sequences. Uracilexcision based cloning is ligase and sequence independent and allows seamless fusion of multiple dna sequences in simple onetube reactions, with higher accuracy than overlapping pcr. Finnzymes phusion highfidelity dna polymerase offers extreme performance for all pcr applications.
The pcr products generated using phusion dna polymerase have blunt ends. Amplification of a short dna stretch by repeated cycles of in vitro dna polymerization science, vol 239, issue 4839, 487491. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols. 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. Only template and primers need to be added by the user. However, it turned out that the allelespecific primers were not that specific at all. This kit uses the highly processive phusion hot start highfidelity. In order to perform pcr, one must know at least a portion of the sequence of the target dna molecule that has to be copied. If tacloning is preferred, then dna should be purified prior to aaddition, as phusion dna polymerase will degrade any overhangs generated.
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