{"product_id":"neb-m0515s","title":"New England Biolabs, M0515S, Q5U® Hot Start High-Fidelity DNA Polymerase","description":"Q5U Hot Start High-Fidelity DNA Polymerase is a modified version of Q5  \u003cb\u003eRelated Categories\u003c\/b\u003e Analysis of DNA Methylation by Bisulfite Conversion,, DNA Methylation Analysis,, PCR, qPCR \u0026amp; Amplification Technologies  \u003cb\u003eApplications\u003c\/b\u003e Applications of USER® and Thermolabile USER II Enzymes,, Bisulfite Sequencing,, USER, ®, Cloning,  \u003cb\u003eFAQ\u003c\/b\u003e Q: How do I enable carryover contamination prevention with Q5U? A: To prevent carryover contamination, dUTP (NEB #N0459) and Antartic Themolabile UDG (NEB #M0372) can be added to the reaction. For best results, we recommend adding dUTP at a final concentration of 200 μM. dTTP can be fully replaced by dUTP and for many targets, no reduction in yield is observed. For UDG activation, a 10 minute, 25°C incubation step should be added before the initial denaturation step. Typical cycling parameters can be used thereafter. Q: Can I use Q5U Hot Start High-Fidelity DNA Polymerase to amplify bisulfite-treated DNA? A: Yes. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 High-Fidelity DNA Polymerase which efficiently amplifies uracil-containing templates. The optimal cycling conditions for bisulfite-treated DNA are different from other applications. Please see Section 2 of the protocol for more details. Q: What are the general recommendations for designing primers for bisulfite-treated\/deaminated DNA? A: We recommend designing long (~26-35) oligonucleotide primers to amplify bisulfite treated\/deaminated DNA. Because bisulfite DNA is often damaged, it is most helpful to design targets between 150-500 bp. Note that since the DNA strands are no longer complementary after bisulfite-treatment\/deamination, an individual primer set will only amplify one strand of the target sequence. The first primer should be designed to anneal to the converted target sequence. The second primer should be designed to anneal to the extension product of the first primer, not the opposite template strand, as would be the case in traditional PCR. Primers with higher annealing temperatures (\u0026gt;60°C as determined by the NEB Tm calculator) are recommended for optimal performance. If needed, include as many guanines as possible in the priming region or add additional bases to increase the Tm. Longer primers will also increase specificity. Ideally, CpG sites should be avoided; if essential, include them on the 5′-end of the primer and have them synthesized with a mixed base (Y= C\/T, R= G\/A) at the cytosine position. Computer programs such as MethPrimer, methBLAST or BiSearch can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction. Q: Can I use Q5U Hot Start High-Fidelity DNA Polymerase for USER® cloning methods? A: Yes. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 High-Fidelity DNA polymerase which efficiently amplifies uracil-containing templates and primers. For USER cloning methods, target DNA molecules and cloning vector are generated by PCR with 8-12 bases of homology between two fragments. PCR primers start with a 5′ A and contain a single deoxyuracil residue (dU) flanking the 3′ end of the homology region, and can be designed to accommodate multiple-fragment assembly, nucleotide substitutions, insertions and\/or deletions. We recommend using the GeneDesign software to design primers for USER junctions. The best results are typically seen when using each primer at a final concentration of 0.5 µM. In comparison to Taq-based enzymes, Q5U Hot Start High-Fidelity DNA Polymerase makes significantly fewer errors, streamlining any cloning protocol (for example, see data below) USER cloning was performed to ligate a 3 kb lacZ gene into a pET21a vector backbone with Q5U Hot Start High-Fidelity DNA Polymerase and Hot Start Taq DNA polymerase. Reactions containing Q5U resulted in a higher assembly efficiency and better accuracy as evidenced by the total number of colonies obtained (A) and percentage of blue colonies (B). Accuracy was further examined using Sanger sequencing which revealed a number of point mutations in the blue colonies obtained from the Taq assembly and no mutations from the 4 clones sequenced from the Q5U experiments (C). The “X”s indicate the number of additional mutations found in the clones produced with Taq, consistent with previous results demonstrating the high mutation tolerance of lacZ. (Barnes, W. M. (1992) Gene, 112, 29-35.) Q: Can I use Q5U Hot Start High-Fidelity DNA Polymerase to amplify FFPE DNA? A: Yes. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 DNA polymerase that can amplify a number of modified bases that are typically present in damaged templates. Successful amplification of FFPE derived materials will be substrate and target specific. 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 \u0026amp; 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 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 \u0026lt;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 \u0026lt;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: My results are not as expected. Where can I find troubleshooting help? A: Nonspecific amplification, no amplification, wrong product size Curious result? Consult our PCR Troubleshooting Guide after your reaction to identify potential causes of unexpected results and solutions. More details on reaction conditions and setup optimization can be found in our Guidelines for PCR Optimization with Thermophilic DNA Polymerases and this blog post. Technical Support is always happy to work with you to troubleshoot your PCR. If you would like assistance, you can: Email us at info@neb.com Call Technical Support at (800)-0632-7799, available Monday through Friday, 9:00AM - 6:00PM EST Fill out this webform Failure to amplify a target greater than 5 kb If you are struggling to amplify a target that is greater than 5 kb, try some of these tips: We recommend using Q5®, Phusion®, or LongAmp® polymerases If using Q5, try decreasing the final primer concentration to 150-300nM Stand-alone enzyme + buffer formulations allow more flexibility in reaction optimization than master mixes Use more template Treat the purified template gently as not to shear it Optimize enzyme concentration by testing a titration of enzyme in the reaction (0.25-2 units\/50μl reactions) Increase the number of cycles Lengthen extension time to 40s\/kb Smearing on an agarose gel When PCR conditions are not optimal, a smear or high level of background is often observed. Try one or more of the following suggestions: Use less enzyme Decrease the extension temperature to 3°C below the extension temperature recommended by the specific product protocol For example, the OneTaq® protocol recommends a 68°C extension temperature; try 65°C. Raise the annealing temperature Try 2-step cycling protocols If there is an illuminated halo around the well in addition to smearing from the well, use less template. Q: What are the differences between the numerous Q5® Polymerase products available? A: Stand-Alone Enzyme formulations (NEB #M0491, #M0493) are recommended for flexible PCR setup and for high GC content templates (by adding the High GC Enhancer to the reaction). Master Mix formulations (NEB #M0492, #M0494) offer the most convenience by containing enzyme, Mg2+, dNTPs, and all necessary buffer components to support robust amplification - only template and primers need to be added. Hot Start formulations (NEB #M0493, #M0494) inhibit the activity of the enzyme, allowing for convenient room temperature reaction set up, and are suitable for all PCR applications requiring greater accuracy, high specificity or the amplification of difficult or long targets. For more information on Hot Start technology, click here. NEBNext® Q5 formulations (NEB #M0541, #M0543, #M0544) have been specifically optimized to limit GC bias during the amplification of NGS libraries. M0544 is the suggested master mix for NGS applications. The Q5 Blood Direct 2X Master Mix (NEB #M0500) is specially formulated for blood samples without the need for a purification step. It is a Hot Start enzyme. Q5 polymerase cannot read through or incorporate uracil, but Q5U® Hot Start High-Fidelity DNA Polymerase (NEB #M0515) can. Use it with uracil-containing, bisulfite-converted, deaminated, or damaged DNA templates. Carryover contamination prevention can be utilized with Q5U and dUTP\/dTTP. It is a Hot Start enzyme. Q: When and how should I use the Q5® High GC Enhancer? A: The Q5 High GC Enhancer is an additive that should be used when dealing with particularly difficult or high GC templates, but can be inhibitory when using high AT content templates. The stand-alone Q5 enzyme can cover a wider range of GC content (up to 80%) with the addition of the GC enhancer. Q5 High GC Enhancer is a supplemental reagent to Q5 enzyme and buffer formulations (M0491 and M0493) and should not be used alone. *This Enhancer is not a stand-alone buffer and should not be used on its own. Additionally, it should not be added to any Q5 Master Mixes (M0492, M0494, M0500, E0555). Q5U does not benefit from the Q5 High GC Enhancer and we do not recommend using it. The addition of common PCR additives, such as up to 2% DMSO, may improve amplification of certain difficult or long targets. It is often not necessary to alter the annealing temperature of your reaction after adding Q5 High GC Enhancer. We recommend using our Tm calculator to determine the annealing temperature of your PCR. Learn More Use of the Q5 High GC Enhancer often lowers the effective range of temperatures at which specific amplification can be observed by reducing complex template secondary structures, which can increase the amplification of your target DNA and improve your yield of hard to amplify products, such as GC rich templates. Generally, PCR additives usually work one of two ways: By reducing secondary DNA structures, thus increasing the amplification of your target DNA Secondary DNA structures can be destabilized by additives that bind to the minor and major grooves of DNA and affecting hydrogen bonding of the duplex. Secondary structures include the double helix (increased hydrogen bonding due to increased GC content) and stem-loop structures (hairpins or bulging nucleotides that reduce hybridization) By reducing non-specific priming and thus reducing the amplification of off-target DNA. Q: Can I use Q5 Reaction Buffer with this enzyme? A: The 5X Q5U Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. The 5X Q5U Reaction Buffer contains 2.0 mM Mg++ at a final (1X) concentration. Q: How can I optimize my product yield using Q5U Hot Start High-Fidelity DNA Polymerase? A: Optimizing depends on application and starting material. Specific recommendations can be found in the protocols. General guidelines are below: Perform a temperature gradient to optimize Tm. Unlike with other polymerases, such as Taq, high fidelity amplification can be sensitive to annealing temperatures, where one or two degrees might make the difference between no amplification and successful amplification of a pure product with a high yield. See the data below for an example. Use more template. Sample concentration may be too low. Optimize enzyme concentration by testing a titration of enzyme in the reaction (0.25-2 units\/50 μl reactions) Increase number of cycles Lengthen extension time to 1min\/kb Change the extension temperature-some applications (amplifying bisulfite-treated, deaminated or damaged DNA) may benefit from using a higher extension temperature then recommended (72°C v 68°C) Annealing Temperature Optimization Can Improve Amplification Results High fidelity amplification of PSMB2 as a function of temperature reveals that robust amplification begins at 65.6 degrees, while little to no amplification is observed a couple of degrees lower. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835534725289,"sku":"M0515S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0515s","provider":"Iright","version":"1.0","type":"link"}