New England Biolabs, M0323S, LongAmp® Taq DNA Polymerase

Exceptional performance with long amplicons Related Categories LongAmp® DNA Polymerases Applications Colony PCR,, Long Range PCR,, Specialty 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 75°C. Reaction Conditions 1X LongAmp® Taq Reaction Buffer Pack 1X LongAmp® Taq Reaction Buffer Pack 60 mM Tris-SO4 20 mM (NH4)2SO4 2 mM MgSO4 3% Glycerol 0.06% IGEPAL® CA-630 0.05% Tween® 20 (pH 9.1 @ 25°C) Storage Buffer 10 mM Tris-HCl 100 mM KCl 1 mM DTT 0.1 mM EDTA 0.5% Tween® 20 0.5% IGEPAL® CA-630 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No 5' - 3' Exonuclease Yes 3' - 5' Exonuclease Yes Strand Displacement + Unit Assay Conditions 1X ThermoPol ® Reaction Buffer, 200 µM dNTPs including [ 3 H]-dTTP and 15 nM primed M13 DNA. FAQ Q: Is Monarch-purified gDNA suitable for long-range PCR? A: Yes. The large fragment size of Monarch-purified gDNA makes it excellent starting material for long range PCR approaches. For long range PCR, we recommend LongAmp TaqDNA Polymerase (NEB #M0323), LongAmp Taq 2x Master Mix (NEB #M0287) or LongAmp Hot Start Taq Polymerase (NEB #M0534). Genomic DNA purified with the Monarch Spin gDNA Extraction Kitis high quality and suitable for sensitive applications like long range PCR and qPCR A. Amplification reactions were set up with primer pairs specific for 6, 8, 10, 12, 16, 20 kb amplicons from human DNA. LongAmp® Hot Start Taq 2x Master Mix (NEB #M0533) was used and 25 ng template DNA was added to each sample. PCR reactions were carried out on an Applied Biosystems 2720 Thermal Cycler. Monarch-purified genomic DNA isolated from HeLa cells and human blood were compared to commercially available reference DNA from the human cell line NA19240 F11. 10 of 20 μl was loaded on a 1.5% agarose gel, using the 1 kb DNA Ladder (NEB #N3232) as a marker. Results indicated DNA was of high-integrity and suitable for long range PCR. B. Monarch-purified genomic DNA from human whole blood, HeLa cells and mouse tail was diluted to produce a five log range of input template concentrations. The results were generated using primers targeting gHEME (human whole blood) and gREL (HeLa, mouse tail) for qPCR assays with the Luna Universal qPCR Master Mix (NEB #M3003) and cycled on a Bio-Rad® CFX Touch qPCR thermal cycler. Results indicated that DNA is highly pure and free from inhibitors, optimal for qPCR. 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: 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 <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: 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.