Safety information when working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. Desai and pfaffle have reported the cloning of taq dna polymerase into puc18 plasmid 8. With surestart taq dna polymerase, hot start is provided by a modified form of taq2000 dna polymerase, a highly purified recombinant version of taq dna polymerase. Pdf rapid purification of highactivity taq dna polymerase. Pcr protocol for taq dna polymerase with standard taq buffer m0273. It is supplied with 10x standard taq reaction buffer, which is detergent free and designed to be compatible with existing assay systems.
Taq dna polymerase is a thermostable dna polymerase that possesses a 5. Takara ex taq dna polymerase combines the proven performance of takara taq polymerase with the proofreading activity of an efficient 3to5 exonuclease, for highsensitivity, highefficiency pcr reactions. This high fidelity derives in part from an integral 35 proofreading exonuclease activity in vent dna polymerase 1,3. Specification see taq dna polymerase, gmp grade, 5 u. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. Taq polymerase is a thermostable dna polymerase i named after the thermophilic eubacterial microorganism thermus aquaticus, from which it was originally isolated by chien et al. Taq dna polymerase, one of the first thermostable dna polymerases to be discovered, has been typecast as a dnadependent dna polymerase commonly employed for pcr. Takara ex taq hs dna polymerase is the hotstart version of our highperforming takara ex taq polymerase a blend of takara taq and a proofreading exonuclease offering high yield, excellent sensitivity, and fidelity that is 4. Salt dependence of dna binding by thermus aquaticusand. Taq dna polymerase is the most common polymerase used for pcr reactions ii. Applications taq dna polymerase can be used in most applications including the following.
Home perelman school of medicine perelman school of. Taq longamp taq component reaction concentration final pcr. Product information polymer taq dna polymerase of reactions. Serial 10fold dilutions of cloned hbv dna, ranging from 1 pg to 107 pg, were amplified by pcr using primers 1763. A dna polymerase is a member of a family of enzymes that catalyze the synthesis of dna molecules from nucleoside triphosphates, the molecular precursors of dna. Taq dna polymerase, the taq pcr core kit, and the taq pcr master mix kit are intended for molecular biology applications. Invitrogen platinum superfi ii dna polymerase is a hotstart, engineered proofreading dna polymerase. Standard taq mg free reaction buffer pack standard taq reaction buffer pack taq dna polymerase with standard taq buffer materials sold separately magnesium chloride mgcl2 solution notes 1. Taq dna polymerase is the robust standard enzyme for the amplification of dna fragments up to 3 kb in pcr, lyo ready formulation for preparation of dried amplification mixes. Taq dna polymerase is a thermostable dna polymerase isolated from an e. Taq dna polymerase is the most common polymerase used for pcr reactions applications taq dna polymerase can be used in most applications including the following. High fidelity is provided by a mixture of platinum taq dna polymerase and the proofreading 3.
Just as with taq dna polymerase, platinum taq dna polymerase has a nontemplatedependent terminal transferase activity that adds a 3. High concentrated, glycerol free solution, ideal for preparation of drieddown amplification mixtures. Contamination and sensitivity issues with a realtime universal. The modified purification method described here is quite simple, however it is important to note that factors such as the bacterial strain used, induction time and protein concentration during isolation have been optimized. Use of taq polymerase as model system is a good way to introduce ip patent system, licensing issues, how patents are. Taq dna polymerase thermostable enzyme used to copy dna free nucleotides the building blocks of dna thermal cycler a. Datasheet for taq dna polymerase with standard taq mgfree. Dna polymerase i or pol i is an enzyme that participates in the process of prokaryotic dna. Dna taq polymerase taq is effective at distinguishing between a match and a mismatch at the 3 end of a pcr primer. Hot diamond taq dna polymerase specification sheet reference. As pcr progresses, the dna generated is used as a template for replication. The polymerase chain reaction pcr is arguably the most powerful laboratory technique ever. It is supplied with 10x standard taq mg free reaction buffer and mgcl 2. It derives its name from one of its key components, a dna polymerase used to amplify a piece of dna by in vitro enzymatic replication.
Taq polymerase also led to the invention of the pcr machine. It is suitable for most general pcr amplification procedures. The enzyme catalyzes 53 synthesis of dna, has no detectable 35 exonuclease proofreading activity and possesses low 53 exonuclease activity. During this process, dna polymerase reads the existing dna strands to create two new. Antibodymediated hotstart gives lower background, higher specificity, and allows. There are three main patents relating to the pcr process and taq dna polymerase. To perform manual hotstart pcr, reactions are assembled on ice or at room.
Oneenzyme reverse transcription qpcr using taq dna. It is especially useful in demanding multiplex assays. The hot start property of the enzyme is conferred by thermolabile monoclonal antibodies that render taq dna polymerase inactive until the initial pcr denaturation step, thus preventing the extention of. This method is based on selective lysis and washing of red blood cells rbc with the roche specimen washing solution rsws and heat andor heatdetergent hd treatment of the separated pbls, to free dna for amplification with taq polymerase. A simple and efficient method for extraction of taq dna polymerase. For medical diagnostics, please use the taq i032 references. The enzyme consists of a single polypeptide with a molecular weight of 94 kda, and will synthesize dna products having da overhangs on the 3 ends. The longamp taq dna polymerase offers two fold higher fidelity than taq. Characterization of a dna polymerase from the hyperthermophile. The recombinant taq dna polymerase shows identical characteristics to native taq from thermus aquaticus. Mechanism of taq dna polymerase inhibition by fullerene derivatives. Longamp taq dna polymerase offers two fold higher fidelity than taq dna polymerase alone.
Empiricals integrity highfidelity dna polymerasetm contains an integral 35 proofreading capability that increases the fidelity of integrity to over 6x of taq dna polymerase. Hot diamond taq dna polymerase specification sheet. Taq full dna polymerase provides the best yield and greatest sensitivity of any taq derived polymerase. Allele or mutationspecific amplification is achieved using arms. Ex taq polymerase has a higher fidelity than standard taq with a. Taq full dna polymerase is a highquality, fulllength recombinant version of the thermus aquaticus strain yt1 dna polymerase engelke et al. Taq pol i from thermus aquaticus taq greatly improves the. Invitrogen platinum taq dna polymerase is a convenient and reliable hot start thermostable dna polymerase for pcr that provides enhanced specificity over that of taq dna polymerase. Isolated from the thermophilic bacterium thermus aquaticus, taq dna polymerase is one of the bestknown thermostable dna polymerases used in pcr amplification of dna targets. Highlevel expression and purification of dna and dnase free taq dna polymerase. A neb onetaq hot start dna polymerase, b qiagen fast cycling pcr kit, c roche. It is supplied with 10x thermopol reaction buffer, which contains a nonionic detergent to increase enzyme stability during longer incubations. Sep, 2012 protocol for taq 2x master mix m0270 overview. Two molecular biology techniques that depend on the properties of dna polymerase the.
Pcr machine a specialized machine that rapidly heats and cools the samples. These products are not intended for the diagnosis, prevention, or treatment of a disease. Taq dna polymerase comes with the choice of an optimized 10. Datasheet for taq dna polymerase with standard taq buffer neb. Taq dna polymerase extends a dna template at approximately 2000 nucleotidesminute, so it is recommended that 4560 seconds of extension time be provided per cycle. Other reports are also available regarding the cloning of this gene 6, 9. However, taq polymerase belongs to the same dna polymerase superfamily as the molony murine leukemia virus reverse transcriptase and has. Taq pol, with their binding finally, site 7 was excluded from md simulations due to the energies eb and population popa lack. Appropriate extension temperatures range from 6672c. To perform pcr, the template is mixed with primers, taq polymerase and nucleotides. Switching the activity of taq polymerase using clamplike triplex. Establishing the detection limits of the pcr technique with recombinant hbv dna.
Taq dna polymerase is a thermostable enzyme that synthesizes dna from singlestranded templates in the presence of dntps and a primer. One unit taq dna polymerase is defined as the amount of enzyme that. Using this strategy, taq polymerase activity can be modulated by reconfiguring the triplex structure. Source hot diamond taq is a highly thermostable enzyme produced and. Kd, and which thus assumes effective equality between the free polym. Without the development of ultrapure taq dna polymerase, ultrapure rea.
Taq dna polymerase, native thermo fisher scientific. Dna damage blocks dna polymerase progression and increases miscoding. Therefore, the native taq dna polymerase is often preferred for amplification of bacterial. Platinum taq dna polymerase, high fidelity, is ideal for amplification of dna fragments when high yields and robust amplification are required. In the absence of adequate free magnesium, taq dna polymerase is inactive. Quickload taq 2x master mix standard taq mg free reaction buffer pack standard taq reaction buffer pack taq 2x master mix taq 5x master mix taq dna polymerase with standard taq mg free buffer taq dna polymerase with standard taq buffer taq dna polymerase with thermopol buffer taq pcr kit thermopol reaction buffer pack thermopol ii. The recombinant enzyme is a cloned version that is purified from e. Surestart taq can be activated by adding an initial step of. Integrity is extremely stable at high temperatures and is free of contaminating endonucleases. Exonuclease active site new strand extension proceeds along the template strand at the 3 end of the newly synthesized strand. To evaluate the thermostability we tested the activity of taq polymerase at three different temperatures. The fidelity of vent dna polymerase is 515fold higher than that observed for taq dna polymerase 1,2. A wide range of pcr products can be generated, up to 30 kb from lambda dna or from human genomic dna.
These enzymes are essential for dna replication and usually work in groups to create two identical dna duplexes from a single original dna duplex. Antibodymediated hotstart gives lower background, higher specificity, and allows room temperature reaction assembly. Pcr amplification an introduction to pcr methods promega. For amplicons taq dna polymerase compared to taq dna polymerase. This sets in motion a chain reaction in which the dna template is exponentially amplified. Surestart taq dna polymerase remains inactive until stringent temperatures e.
When the polymerase recognizes an error, the mismatched base is transferred to the exonuclease active site and the base is excised. For standard and specialized pcr applications with minimal optimization pdf kb. The modified purification method described here is quite simple, however it is important to note that factors such as the bacterial strain used, induction time and protein concentration during isolation have been. It is frequently used in the polymerase chain reaction pcr, a method for greatly amplifying the quantity of short segments of dna. It has a 53 dna polymerase activity and a 53 exonuclease activity see figure. We have devised a method of sample processing applicable to clinical blood samples to be tested by pcr. Titanium taq sp dna polymerase is a highly sensitive, robust, hotstart enzyme for use in pcr applications. Taq dna polymerase is an 832amino acid protein with an inferred molecular weight of 93,920 and a specific activity of 292,000 units mg.
The enzyme consists of a single polypeptide with a molecular weight of 94 kda. The taq dna polymerase gene is isolated from thermus aquaticus yt1 and expressed in li. Takara ex taq dna polymerase combines the proven performance of takara taq polymerase with the proofreading activity of an efficient 3to5 exonuclease, for highsensitivity, highefficiency pcr. The longamp hot start taq dna polymerase offers twofold higher fidelity than taq.
The ampli ed taq polymerase gene was digested with. After stainsall sigmaaldrich or eb sangon biotech staining. Taq polymerase 200units 40l units 200l 5000units 1ml rubybuffer 1. The latter option may be necessary to determine optimal conditions for amplification. Buffer, which is detergent free and designed to be compatible with existing assay systems. Platinum ii taq hotstart dna polymerase thermo fisher. Thermo scientific native taq dna polymerase is a highly thermostable dna polymerase of the thermophilic bacterium thermus aquaticus. The method described here is derived from that of engelke et al. It contains the same titanium taq dna polymerase and taqstart antibody as our standard titanium taq products, but with a modified, license free buffer formulation that is suitable for commercial and oem use, including molecular. Taq 2x master mix should be used at a 1x concentration with dna template and primers in a total reaction volume of 25 or 50. Ex taq dna polymerase hot start version takara bio. Because dna from a wide range of sources can be amplified, the technique has been applied to many fields. Polymerase domain polymerase domain template strand polymerase active site 5.
The polymerase chain reaction pcr is a technique widely used in molecular biology. Pdf optimization of taq dna polymerase enzyme expression in. Pdf in the present study, we optimized the experimental conditions using. Pdf production and evaluation of taq dna polymerase. Dna damage reduces taq dna polymerase fidelity and pcr. Its fidelity is 300x that of taq dna polymerase, and its buffer is specially formulated for universal annealing all primers annealing at 60c.
Pcr is used to diagnose genetic disease and to detect low levels of viral infection. Detergent free polymerases ep 2374874 published october, 2011 chimeric dna polymerase wo 2006010887 a1. The native enzyme is purified from thermus aquaticus yt1. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Toll free in the usa 8003569526 telephone 6082744330 usage information b. Water, nuclease free to 50 l 10x pcr buffer, mg 5 l 1x 50 mm mgcl 2 1. It can also be used for longrange pcr up to 20 kb from genomic dna templates and up to 30 kb from lambda dna templates. This protocol, using taq dna polymerase see unit 3. Buffer choice we recommend using the 5x green gotaq reaction buffer in any amplification reaction. Taq polymerase is an antibody inactivated hot start enzyme designed to minimize nonspecific amplification while increasing target yield. Platinum ii taq hotstart dna polymerase enables cycling of shorter and longer amplicons together. Use of taq polymerase as model system is a good way to introduce ip patent system, licensing issues, how patents are structured, and recent changes to patent system. L reactions using platinum ii taq hotstart dna polymerase or other hotstart dna polymerases.
Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. Taq i033 eurogentec products are sold for research or laboratory use only and are not to be administrated to humans or used for medical diagnostics. Polymerase activity was found in the flowthrough fractions and was free of the. In the atailing reaction, taq polymerase adds a single a to the 3 ends of the blunted dna in a templateindependent fashion. The following guidelines are provided to ensure successful pcr using new england biolabs taq 2x master mix. Polymerase chain reaction wikipedia, the free encyclopedia. Taq dna polymerase is a very important enzyme for molecular biological studies such as dna amplification. Using platinum taq dna polymerase the hotstart property of platinum taq dna polymerase allows for convenient reaction assembly at room temperature. Highlevel expression, purification, and thermus aquatlcus dna. The halflife time rapidly increases with only a few degrees of temperature drop.
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