New England Biolabs, M0535L, Phusion™ Hot Start Flex DNA Polymerase

Related Categories Phusion™ High-Fidelity DNA Polymerases Applications Hot Start PCR,, Long Range PCR,, Fast PCR, Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C. Storage Buffer 20 mM Tris-HCl 100 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol 1X stabilizers pH 7.4 @ 25°C Heat Inactivation No 5' - 3' Exonuclease No Unit Assay Conditions 25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl 2 , 1 mM β-mercaptoethanol, 200 μM dNTPs including [ 3 H]- dTTP and 15 nM primed M13 DNA. FAQ Q: What are the differences between the various available Phusion™ products? A: Stand-alone enzyme formats (NEB #M0530, #M0535, and #E0553) of Phusion polymerase provide the most flexibility for reaction optimization, and are supplied with two buffers: HF and GC. HF buffer is the default buffer. GC buffer should be used when HF buffer fails or when templates contain high GC content or secondary structures. Both HF Buffer Pack (NEB #B0518) and GC Buffer Pack (NEB #B0519) are available for individual purchase. We offer Phusion master mix formulations premixed with the HF buffer (NEB #M0531) or GC buffer (NEB #M0532) and dNTPs- just add your template and primers. Hot Start versions of the stand-alone Phusion polymerase (NEB #M0535) and master mix (NEB #M0536) permit room temperature setup and may decrease nonspecific amplification during early ramping. To learn more about Hot Start enzyme activity control, click here. The Phusion High-Fidelity PCR Kit (NEB #E0553) contains all reagents needed for PCR, including dNTPs, DMSO, a positive control, and DNA ladder for subsequent gel electrophoresis. 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 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 does exonuclease activity mean for a DNA polymerase? A: Polymerases can possess up to two types of exonuclease activity: 5´- 3´ exonuclease activity will digest nucleotides in the 5´ to 3´ direction on a template ahead of polymerase activity. Taq polymerase possesses a specific type of 5´-3´ exonuclease activity called 5' flap endonuclease activity, which catalyzes the cleavage of 5´ DNA flaps from a DNA duplex that has a single-stranded 5´ overhang on one of the strands. 3´ - 5´ exonuclease activity, AKA proofreading activity, digests nucleotides with 3´ hydroxyl groups from the 3´ to 5´ direction. This includes correction of mismatched base pairs and terminal 3´ digestion. Some polymerases possess one or both of these types of exonuclease activities. In the presence of dNTPs, polymerization activity is higher than exonuclease activity. Some polymerases possess neither of these activities. Learn More What does it mean when a polymerase has 5´- 3´ exonuclease activity? When polymerases with 5´- 3´ exonuclease activity encounter a downstream nucleotide on the same strand, the phosphodiester bonds of the encountered strand are hydrolyzed (digested) ahead of polymerization. Taq polymerase possesses a specific type of 5´- 3´ exonuclease activity called 5´ flap endonuclease activity, which catalyzes the cleavage of 5´ DNA flaps from a DNA duplex that has a single-stranded 5' overhang on one of the strands. When Taq polymerase encounters such a flap structure, such as the fluorophore on a qPCR hydrolysis probe, it sequentially digests the phosphodiester bonds between nucleotides in the 5´- 3´ direction ahead of polymerization. The relationship between 3´ - 5´ exonuclease (proofreading) activity and PCR product length. DNA polymerases ensure accurate replication using a series of molecular checkpoints at the site of nucleotide incorporation and beyond. During nucleotide addition, the correct incoming nucleotide is positioned for a productive alignment of catalytic groups, ensuring efficient incorporation. This alignment for catalysis is sensitive to distortions in position caused by incorrect Watson-Crick base pairing, allowing for kinetic stalling at incorrect or non-cognate base pairs. Polymerases that possess proofreading activity “check” whether or not the correct nucleotide has been inserted into the template. If a mismatch is detected, the DNA is transferred from the polymerization domain to the N-terminal 3´ - 5´ exonuclease domain of the polymerase. The incorrectly incorporated nucleotide is excised, DNA moves back into the polymerization domain, and copying can resume. The absence of 3´ - 5´ exonuclease (proofreading) activity may have ramifications other than fidelity in PCR. The lack of proofreading activity in Taq DNA Polymerase has been proposed to limit the maximum amplicon size possible. Generally, Taq performs best when amplifying DNA fragments < 2 kb but can work with fragments up to 3 – 4 kb. When kept to this amplicon size, Taq is a robust, easily optimized enzyme. However, with products above ~3 kb it quickly drops in effectiveness. During PCR, Taq will misincorporate nucleotides and produce mismatches, making it prone to stalling and more likely to dissociate before extending the full-length product. The combination of a certain amplicon size and polymerase error rate can result in accumulation of enough mismatched 3´ ends to effectively inhibit the PCR process. These mismatched 3´ ends are particularly problematic for Taq because it lacks the 3´→ 5´ exonuclease activity to remove them. By mixing in a small amount of a polymerase with proofreading exonuclease activity, such as Deep Vent® DNA Polymerase, amplification of fragments ≥ 20 kb can be achieved (see LongAmp® DNA Polymerase). Since the vast majority of the enzyme in the blend is Taq DNA Polymerase, it is probably doing the bulk of the primer extension, with the proofreading Deep Vent DNA Polymerase removing the inhibitory 3´ mismatches generated by Taq. To learn more about the anatomy of a polymerase and how structure relates to function, see this Feature Article. How to block exonuclease activity during PCR? Add phosphorothioate linkages to primers. A phosphorothioate (pt) bond is a phosphodiester linkage where one of the two non-bridging oxygens has been replaced by a sulfur atom, which inhibits nuclease phosphodiesterase activity. Chemically, the substitution of oxygen with sulfur does not dramatically change the reactivity of the bond, and pt-containing polynucleotides can still function in many enzymatic reactions. In most contexts, 2-3 pt bonds at the 3´ end are sufficient to block polymerase 3´-5´ exonuclease activity. Q: Does Phusion™ High-Fidelity DNA Polymerase exhibit a strand displacement activity? A: No. Phusion High-Fidelity DNA Polymerase has an extremely weak tendency to displace downstream non-templated sequence so it is not considered a property of the enzyme. 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: 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: My template is GC-rich or supercoiled. How can I optimize my product yield using Phusion™ High-Fidelity DNA Polymerase? A: * Add 2-8% DMSO in the final reaction. This often requires a reduction in annealing temperature by 2-5°C. * Optimize initial denaturation time. For most templates the optimal initial denaturation time is 30 seconds, but up to 3 minutes can be used for complex templates. * If HF buffer has failed, try using GC buffer. GC buffer allows better amplification of GC-rich templates, but slightly lowers fidelity. Alternately, try Q5® High-Fidelity DNA Polymerase (NEB# M0491) and include the Q5 High GC Enhancer additive. 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.