M0480 FAQs for OneTaq® DNA Polymerase, Routine PCR, Intl, NEB
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OneTaq® DNA Polymerase FAQ

Q1: How should I set up a PCR reaction using OneTaq® DNA Polymerase?
Q2: Can I use my regular Taq-based cycling conditions for OneTaq® DNA Polymerase based products?
Q3: Which buffer should I use?
Q4: Is OneTaq® DNA Polymerase active in other Taq reaction buffers?
Q5: Can I use OneTaq® DNA Polymerase in "hot start" PCR to help with specificity or primer dimer problems?
Q6: When should I add the High GC Enhancer?
Q7: Can OneTaq® DNA Polymerase be used in colony PCR?
Q8: What type of DNA ends result from a primer extension reaction or a PCR using OneTaq® DNA Polymerase?
Q9: How long a product can be made by OneTaq® DNA Polymerase?
Q10: Can OneTaq® DNA Polymerase be used with uracil-containing primers or bisulfite treated DNA?
Q11: What is the fidelity of OneTaq® DNA Polymerase?
Q12: Where can I find help troubleshooting my PCR?
Q13: How should I determine an appropriate annealing temperature for my reaction?
Q14: Which buffer should I use if I want to control the level of magnesium in the reaction?
Q15: How is OneTaq different from LongAmp Taq?

Q1: How should I set up a PCR reaction using OneTaq® DNA Polymerase?

A1: The general guidelines for a 50 µl reaction are:
1.25 units OneTaq® DNA Polymerase
200 μM each dNTP
0.2 μM each primer
2-50 pg plasmid or 50-500 ng genomic template
1 X Standard Buffer (GC Reaction Buffer can be used for GC-rich amplicons)
Denature at 94°C
Extend 1 minute/kb at 68°C


Q2: Can I use my regular Taq-based cycling conditions for OneTaq® DNA Polymerase based products?

A2: OneTaq® DNA Polymerase performs well in Taq-based protocols. Although protocols can vary, we have found that 68°C is a better default extension temperature than 72°C.


Q3: Which buffer should I use?

A3: For most routine applications, we recommend using the OneTaq® Standard Reaction Buffer. For difficult/GC rich amplicons, the OneTaq® GC Reaction Buffer can improve specificity and/or yield. Please see the Buffer Selection Table for detailed guidance.


Q4: Is OneTaq® DNA Polymerase active in other Taq reaction buffers?

A4: The OneTaq® Standard and GC Reaction Buffers have been optimized for robust performance over a wide range of target amplicons. For routine/easy targets, other Taq Reaction Buffers can be used successfully, however for high AT or GC amplicons they typically result in decreased reaction specificity and/or suboptimal yields.


Q5: Can I use OneTaq® DNA Polymerase in "hot start" PCR to help with specificity or primer dimer problems?

A5: For "hot start" PCR,
OneTaq® Hot Start DNA Polymerase is the preferred choice.


Q6: When should I add the High GC Enhancer?

A6: For particularly difficult or high GC amplicons, the enhancer can be added to the GC Reaction Buffer to improve specificity and/or yield. The enhancer is not a buffer and should not be used alone. Final concentration of the enhancer in the amplification reaction should be between 10-20%. In our experience, it has limited effects when added to the OneTaq® Standard Reaction Buffer but when added to the OneTaq® GC Reaction Buffer, can improve yields when other conditions have failed.


Q7: Can OneTaq® DNA Polymerase be used in colony PCR?

A7: Yes, it is a good choice for colony PCR. We recommend an initial 2-5 min denaturation step to lyse cells.


Q8: What type of DNA ends result from a primer extension reaction or a PCR using OneTaq® DNA Polymerase?

A8: A sufficient proportion of PCR products generated using OneTaq® DNA Polymerase contain dA overhangs at the 3´end to enable ligation to dT/dU-overhang vectors.


Q9: How long a product can be made by OneTaq® DNA Polymerase?

A9: The presence of a proofreading polymerase in the formulation allows slightly longer amplicons to be made by OneTaq® DNA polymerase than by Taq DNA polymerase alone. From complex genomic DNA templates, we can routinely amplify fragments to 6 kb.


Q10: Can OneTaq® DNA Polymerase be used with uracil-containing primers or bisulfite treated DNA?

A10: The presence of an archaeal polymerase does not prevent OneTaq® DNA polymerase from using dU-containing DNA as a substrate. OneTaq DNA Polymerase is compatible with USER cloning methods and bisulfite treated DNA. For protocols employing bisulfite treated DNA that require a hot start enzyme, we recommend OneTaq® Hot Start DNA Polymerase.


Q11: What is the fidelity of OneTaq® DNA Polymerase?

A11: The presence of a proofreading polymerase increases the fidelity of OneTaq® DNA polymerase to approximately 2-fold greater than Taq DNA polymerase alone.


Q12: Where can I find help troubleshooting my PCR?

A12: Please find information about
common PCR problems here.


Q13: How should I determine an appropriate annealing temperature for my reaction?

A13: Please use
NEB’s Tm Calculator to determine the appropriate annealing temperature for your primer pair and polymerase/buffer of interest.


Q14: Which buffer should I use if I want to control the level of magnesium in the reaction?

A14: For complete control of magnesium levels,
OneTaq® (Mg-free) Standard Reaction Buffer and OneTaq® (Mg-free) GC Reaction Buffer are also available.


Q15: How is OneTaq different from LongAmp Taq?

A15: Both OneTaq and LongAmp Taq DNA polymerases are blends of Taq and Deep Vent DNA Polymerases. Both enzyme blends take advantage of the observation that the addition of a proofreading (exo+) polymerase to Taq increases performance [Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA, 91, 2216-2220], however the concentration and ratio of the enzymes is different in each blend. For LongAmp Taq, the supplied buffer, enzyme concentrations and ratio have been adjusted to favor robust amplification of moderate to very long products (≤ 30 kb lambda or human genomic). OneTaq and the OneTaq, along with the OneTaq Standard Buffer, GC Buffer and High GC Enhancer, have all been formulated for robust performance of short to moderate length products (≤ 6kb genomic) across very low to very high GC content (~15-85%).