New England Biolabs, M0495S, Hot Start Taq DNA Polymerase

The industry standard with hot start performance Related Categories Taq, DNA Polymerase Applications Colony PCR,, Multiplex PCR,, Specialty PCR, Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 15 nmol of dNTP into acid insoluble material in 30 minutes at 75°C. Reaction Conditions 1X Standard Taq Reaction Buffer Pack 1X Standard Taq Reaction Buffer Pack 10 mM Tris-HCl 50 mM KCl 1.5 mM MgCl2 (pH 8.3 @ 25°C) Storage Buffer 10 mM Tris-HCl 100 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 1X stabilizers pH 7.4 @ 25°C Heat Inactivation No Unit Assay Conditions 1X ThermoPol® Reaction Buffer, 200 µM dNTPs including [ 3 H]-dTTP and 15 nM primed M13 DNA. FAQ Q: What ends will my PCR products have? A: APPLICATION POLYMERASE PRODUCTS PCR PRODUCT ENDS High fidelity PCR Q5® polymerases Blunt Phusion® polymerases Blunt Routine & Specialty PCR OneTaq® polymerases 3'A/blunt Taq polymerases 3'A LongAmp® polymerases 3'A/blunt Hemo KlenTaq Polymerase 3'A Isothermal amplification Bst polymerases 3'A Bsu Polymerase 3'A phi29 Polymerase Blunt DNA manipulation T7 DNA Polymerase Blunt E. coli DNA Polymerase I Blunt DNA Polymerase I, Large (Klenow) Fragment Blunt Klenow Fragment (3′-5′ exo-) 3'A T4 DNA Polymerase Blunt Vent® Polymerase Blunt Vent® (exo-) Polymerase 3'A Deep Vent® Polymerase Blunt Deep Vent® (exo-) Polymerase 3'A For more details about our polymerases, including exonuclease activities and applications, please visit our DNA Polymerase Selection Chart. Learn More For more information about exonuclease activity, check out this FAQ. Why do some polymerases blunt and others add a nucleotide? Polymerases that possess proofreading (3´-5´ exonuclease) activity, such as Q5, Phusion, and Deep Vent, will add an untemplated nucleotide to the 3' ends of extended DNA fragments, but the exonuclease activity subsequently removes it. Other polymerases that lack 3´-5´ exonuclease activity (such as Taq and Taq-based polymerases) will add an extra nucleotide to 3´ ends (predominantly, but not exclusively, dA) and leave the untemplated overhang intact. This is why it is important to know which polymerase to use when performing blunt-end or T/A cloning. OneTaq and LongAmp Taq DNA polymerases are optimized blends of Taq (a Family A polymerase) and Deep Vent (a Family B polymerase) DNA Polymerases. The intrinsic polymerase activity of Taq adds a non-templated 3´A, while the 3´–5´ exonuclease activity of Deep Vent increases the fidelity and robustness of Taq, but also blunts PCR products. This is why these products produce a mixture of DNA ends. However, the majority of ends will have a 3'A overhang. Q: What are Hot Start and WarmStart® polymerases and when would I use them? A: When setting up room temperature reactions off ice When non-specific amplification is observed What does Hot Start/ Warm Start mean? Our Hot Start and WarmStart polymerases utilize aptamers that inhibit enzyme activity at room temperature, which discourages the formation of nonspecific products. The presence of these aptamers does not alter core enzyme function. The distinction between Hot Start and WarmStart is that Hot Start enzymes are thermophilic while WarmStart® enzymes are mesophilic; the thermodynamic range of the aptamers for Hot Start and WarmStart enzymes are largely similar. Aptamers are engineered oligonucleotides that bind to a specific target molecule through non-covalent interactions and include specific nucleobase modifications that can improve inhibition profiles and/or reduce unintended side effects. The benefit of aptamer-based inhibition over other Hot Start technologies (like antibodies) is that they do not require an activation step and bind reversibly in a temperature-dependent manner. NEB offers polymerases with aptamers for routine PCR, high-fidelity PCR, isothermal amplification, and reverse transcription. These aptamers can target different enzymatic functions for different polymerases. Learn More Why is it called "Hot Start?" Like many non-proofreading, Family A DNA polymerases, Taq Polymerase possesses the ability to add bases onto the end of ssDNA in a template-independent manner even at room temperature, and can result in the addition of non-specific bases onto the ends of DNA primers in the reaction, enabling off-target hybridization and reduced overall reaction specificity. In contrast, at higher temperatures, nonspecific binding is reduced as annealing becomes more stringent. Early methods to mitigate undesired activity at low temperature focused on the exclusion of key reaction components until the reaction temperature was increased, which could then be spiked into the mixture, triggering the reaction under a more restrictive, high temperature condition. Read our feature article, Using aptamers to control enzyme activities Hot Start Taq and beyond, to see data comparing product formation for enzymes with and without aptamer-based inhibition of activity. Q: How should I determine the appropriate annealing temperature for my reaction? A: The optimal annealing temperature (Ta) for a primer pair can be determined empirically by running a gradient PCR. Please use NEB’s Tm Calculator to determine the initial annealing temperature for your primer pair and the NEB polymerase/buffer to be used. Unlike other calculators, the NEB Tm Calculator takes into consideration buffer components that affect melting temperatures and empirical observations when calculating the optimal annealing temperature. Other online calculators may underestimate the best Q5 polymerase annealing temperature. For more information on using a single (i.e., "universal") annealing temperature, please see our application note: Universal Annealing Temperature in PCR and its Impact on Amplification Results. Learn More Efficient PCR is a dynamic balancing act of chemicals and reactants that promote specific primer interaction with its compliment in the template at the selected annealing temperature. While annealing temperatures are constant values selected by the scientist, melting temperatures between each primer and the template can differ from amplicon to amplicon. Definitions Note: this section specifically discusses annealing of an oligonucleotide primer to a DNA template. During the denaturation step of PCR, high temperature separates template dsDNA into ssDNA, revealing complex nucleotide sequences that permit annealing (binding, hybridization, association) of a complimentary single-stranded oligonucleotide primer at a lower temperature. The annealing temperature (TA) is the temperature used during the primer annealing step of a PCR, which is dependent on primer melting temperature. The melting temperature (TM) of a primer is the temperature at which 50% of the primer is bound to its perfect complement and 50% is free in solution due to dissociation ("melting") from its compliment. Why using the correct annealing temperature is important for successful PCR The annealing temperature of a reaction is usually lower than the melting temperature to ensure primer hybridization to the template. If the annealing temperature is too high, the primer will not anneal to the template and amplification will not proceed. If the annealing temperature is too low, nonspecific binding of the primer(s) to the template or each other (primer dimers) can occur, causing: Increased likelihood of nonspecific product formation. Decreased formation of the intended product due to inefficient reaction conditions. PCR reactants that influence primer melting temperature and reaction annealing temperature Melting temperatures are not constant values in a PCR and are influenced by a number of factors: Primer length and proportion of guanine and cytosine relative to adenine and thymine (% GC content) Dictates the amount of hydrogen bonding between the primer and its compliment. The more hydrogen bonding (higher Tm) of a primer to its template, the more energy needed to break those bonds (higher temperature). Primer concentration The melting temperature of primers in a PCR is determined by the DNA species in molar excess, which should be the primers. Magnesium and dNTPs The free concentration of magnesium ions [Mg2+] determines the melting temperature of a DNA duplex, but magnesium can be sequestered by the reactants and products of the PCR. The positive charge of magnesium chelates the negatively charged phosphates of dNTPs, primers, and ssDNA. Reduction of electrostatic repulsions (between primer and ssDNA phosphates) increases primer Concentration of monovalent cations (Na+, K+) Monovalent cations support DNA duplex stability, similarly to magnesium ions. Monovalent cations and magnesium ions compete for DNA binding. Increasing monovalent cation concentration decreases magnesium binding to DNA. Q: What should the final primer concentration be in my PCR? A: POLYMERASE PRODUCTS RECOMMENDED FINAL PRIMER CONCENTRATION (EACH) FINAL PRIMER CONCENTRATION RANGE (EACH) Q5® Polymerases 500 nM 200-1000 nM Phusion® Polymerases 500 nM 200-1000 nM OneTaq® Polymerases 200 nM 50-1000 nM Taq Polymerases 200 nM 50-1000 nM Hemo KlenTaq 300 nM 50-1000 nM LongAmp® Polymerases 400 nM 50-1000 nM We recommend a final primer concentration of 500 nM when using Q5 and Phusion polymerases due to their high 3´-5´ exonuclease activity. We recommend a final primer concentration of 200 nM when using Taq-based polymerases, including OneTaq and EpiMark®. Please refer to specific protocols on the product pages for details regarding range recommendations. Learn More Archaeal Family B polymerases, such as Q5 and Phusion, possess strong 3´-5´ exonuclease activity, which can digest nucleotides on the 3' end of primers and increase the likelihood of nonspecific amplification. Relative to Family A polymerases like Taq and OneTaq, a higher final primer concentration overcomes these effects and promotes specific product formation. Generally, efficient PCR requires optimal ratios of magnesium, other ions, and primers to promote specificity by stabilizing the negative charges on the phosphate backbone. While the composition of Q5 and Phusion DNA Polymerases are proprietary, their buffers are optimized for using a final primer concentration of 500nM. Q: What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)? A: POLYMERASE PRODUCTS FIDELITY* ERROR RATE PRODUCT ENDS MAX PRODUCT LENGTH** EXTENSION TEMPERATURE MODIFIED NUCLEOTIDE INCORPORATION*** URACIL INCORPORATION 5´-3´ EXONUCLEASE 3´-5´ (PROOFREADING) EXONUCLEASE Q5 Polymerases 280X <0.44 x 10-6 Blunt 20kb simple, 10kb complex 72°C 5mC, 5hmC, 6mA No (except Q5U) - ++++ Phusion Polymerases 39-50X 0.44 x 10-6 Blunt 20kb simple, 10kb complex 72°C 5mC, 5hmC No - ++++ OneTaq Polymerases 2X <140 x10-6 3´A/blunt 6kb 68°C 5mC, 5hmC, biotin, DIG Yes + ++ Taq Polymerases 1X 2.85 x 10^-4 3´A 5kb 68°C 5mC, 5hmC, biotin, DIG Yes + - Hemo KlenTaq nt nt 3´A 2kb 68°C Yes No - LongAmp® Polymerases 2X 3´A/blunt 30kb 65°C No Yes ++ *Fidelity relative to Taq DNA polymerase. We continue to investigate assays to characterize Q5's very low error rate to ensure that we present the most accurate fidelity data possible (Potapov, V, and Ong, J.L. (2017) PloS ONE, 12(1): e0169774). **Simple templates include plasmid, viral and E. coli genomic DNA. Complex templates include plant, human and other mammalian genomic DNA and cDNA. *** For more information, contact Technical Support at info@neb.com For more information on properties to help you select a polymerase for your application, please see our DNA Polymerase Selection Chart. Learn More Fidelity and error rate The fidelity of a DNA polymerase is defined by its ability to accurately replicate a template, while error rate is the rate of misincorporation of an incorrectly matched nucleotide. Fidelity is important for applications in which the DNA sequence must be correct after amplification. To learn more about how fidelity is measured, click here. Product ends and exonuclease activity Check out the "Learn More" section on our PCR Product Ends FAQ and our Exonuclease Activity for DNA Polymerases FAQ. Q: What are the stability and storage requirements for NEB's Taq and OneTaq DNA Polymerases? A: The Taq and OneTaq products listed below should be stored at -20C and have a shelf life of 24 months: M0267 Taq DNA Polymerase with ThermoPol® Buffer M0270 Taq 2X Master Mix M0271 Quick Load Taq 2X MM M0271 Quick-Load® Taq 2X Master Mix M0273 Taq DNA Polymerase with Standard Taq Buffer M0284 Multiplex PCR 5X Master Mix M0285 Taq 5X Master Mix M0320 Taq DNA Polymerase with Standard Taq (Mg-free) Buffer M0495 Hot Start Taq DNA Polymerase M0496 Hot Start Taq 2X Master Mix M0480 OneTaq® DNA Polymerase M0481 OneTaq® Hot Start DNA Polymerase M0482 OneTaq® 2X Master Mix with Standard Buffer M0484 OneTaq® Hot Start 2X Master Mix with Standard Buffer M0485 OneTaq® Hot Start 2X Master Mix with GC Buffer M0486 OneTaq® Quick-Load® 2X Master Mix with Standard Buffer M0488 OneTaq® Hot Start Quick-Load® 2X Master Mix with Standard Buffer M0489 OneTaq® Hot Start Quick-Load® 2X Master Mix with GC Buffer Q: Is this Taq DNA Polymerase product compatible with other NEB Buffers? A: Product name Hot Start TaqDNA Polymerase TaqDNA Polymerase with Standard Buffer Taq DNA Polymerase with Standard Buffer (Mg free) TaqDNA Polymerase with ThermoPol Buffer EpiMark® Hot Start TaqDNA Polymerase Product number M0495 M0273 M0320 M0267 M0490 Supplied buffer Standard Taq Reaction Buffer Standard Taq Reaction Buffer Standard Taq (Mg-free) Reaction Buffer with 25mM MgCl2 ThermoPol® Reaction Buffer EpiMark® Hot Start Taq Supplied buffer composition at 1X (final reaction concentration) 10 mM Tris-HCl 50 mM KCl 1.5 mM MgCl2 (pH 8.3 @ 25°C) 10 mM Tris-HCl 50 mM KCl 1.5 mM MgCl2 pH 8.3@25°C 0 mM Tris-HCl 50 mM KCl pH 8.3@25°C 20 mM Tris-HCl 10 mM (NH4)2SO4 10 mM KCl 2 mM MgSO4 0.1% Triton ® X-100 pH 8.8@25°C 20 mM Tris-HCl 1.8 mM MgCl2 22 mM KCl 0.06% IGEPAL® CA-630 0.05% Tween ® 20 22 mM NH4Cl (pH 8.9 @ 25°C) Other compatible buffers *Use in rCutSmart™ or otherNEBuffers is not recommended* ThermoPol® Reaction Buffer OneTaq ® Standard Reaction Buffer LongAmp® TaqReaction Buffer ThermoPol® Reaction Buffer OneTaq® Standard Reaction Buffer LongAmp® TaqReaction Buffer Standard Taq Reaction Buffer OneTaq® Standard Reaction Buffer LongAmp® Taq Reaction Buffer Standard Taq Reaction Buffer One® Standard Reaction Buffer LongAmp® Taq Reaction Buffer ThermoPol® Reaction Buffer Standard TaqReaction Buffer Q: Can Taq/OneTaq® DNA Polymerases be used for nick translation? A: Yes. This type of exonuclease activity can be used for nick translation and is also a property of DNA Polymerase I (NEB# M0209). Q: How can I optimize my PCR when using Taq DNA Polymerase? A: See usage guide.