New England Biolabs, M0332L, Hemo KlenTaq®

Eliminate DNA extraction and purification Related Categories Other PCR Polymerases Applications Extraction-Free PCR,, Specialty PCR,, PCR Specification Reaction Conditions 1X Hemo KlenTaq® Reaction Buffer Pack 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 No 3' - 5' Exonuclease 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: 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: 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.