Guidelines for PCR Optimization with OneTaq® and OneTaq® Hot Start DNA Polymerases

OneTaq DNA Polymerase (NEB #M0480) is an optimized blend of Taq (NEB #M0273) and Deep Vent® (NEB #M0258) DNA Polymerases for use with routine and difficult PCR experiments. The 3´→ 5´ exonuclease activity of Deep Vent DNA Polymerase increases the fidelity and robust amplification of Taq DNA Polymerase (1).

OneTaq Hot Start DNA Polymerase (NEB #M0481) includes an aptamer-based inhibitor, allowing convenient room temperature reaction set up and decreased interference from primer dimers and secondary products.

The OneTaq Reaction Buffers and High GC Enhancer have been formulated for robust yields with minimal optimization, regardless of a template's GC content.

Guidelines (except where specified, guidelines apply to both OneTaq and OneTaq Hot Start DNA Polymerases)

OneTaq Buffer Recommendations

<50% GC OneTaq Standard Reaction Buffer Adjust annealing temperature, primer/
template concentration, etc., if needed.
50–65% GC OneTaq Standard Reaction Buffer OneTaq GC Reaction Buffer can be
used to enhance performance of difficult
>65% GC OneTaq GC Reaction Buffer OneTaq GC Reaction Buffer with
10–20% OneTaq High GC Enhancer
can be used to enhance performance on
difficult amplicons.

DNA Template

  • Use high quality, purified DNA templates, unless performing colony PCR
  • Approximately 104 copies of target DNA are required to detect product in 25-30 PCR cycles
  • Use 1 pg–10 ng of plasmid or viral templates
  • Use 1 ng–1 µg of genomic templates
  • Higher DNA concentrations decrease amplicon specificity (i.e., extra bands are more likely), particularly when a large number of cycles are employed
  • Use the higher DNA concentrations when fewer cycles are desired (e.g., to increase fidelity)


  • Generally 20–30 nucleotides in length
  • Ideal GC content is 40–60%
  • Space GC residues evenly within the primer (avoid gc rich 3' ends)
  • Calculated melting temperatures (Tm) should be from 45-68°C
  • Primer pairs should have Tms within 5°C of each other
  • Avoid secondary structure (i.e., hairpins) within each primer and potential dimerization between the primers present
  • When engineering restriction sites into the end of primers, 4-6 extra bases should be added 5´ to the site
  • Final concentration of each primer is typically 0.2 µM but can range between 0.05–1 µM
  • Higher concentrations may increase secondary priming and create spurious amplification products
  • Use the NEB Tm Calculator to calculate an appropriate annealing temperature.

Magnesium Concentration

  • 1.5–2.0 mM is optimal for OneTaq DNA Polymerase
  • Optimal concentration depends on template, buffer, DNA and dNTPs (each has the potential to chelate magnesium)
  • If [Mg2+] is too low, no PCR product will be seen
  • If [Mg2+] is too high, undesired PCR products may be seen
  • Optimize by supplementing magnesium concentration in 0.2 increments up to 4 mM

OneTaq DNA Polymerase Concentration

  • OneTaq can be used from 0.25–5 units per 50 µl reaction
  • For amplicons up to 3 kb, we recommend 1.25 units per 50 µl reaction
  • For specialized applications, including 3–6 kb amplicons, 2.5–5 units per 50 µl reaction is recommended

Starting Reactions

  • Assemble all reaction components on ice
  • Add polymerase last
  • Immediately transfer reactions to thermocycler preheated to denaturation temperature (94°C)
OneTaq Hot Start
  • Reactions can be assembled at room temperature
  • Transfer reactions to a room temperature thermocycler and begin cycling protocol

Denaturation Temperature and Duration

  • Initial denaturation at 94°C for 30 seconds to 2 minutes is recommended prior to PCR cycling to fully denature the DNA
  • A longer denaturation of 2–4 minutes is recommended for high GC templates
  • For colony PCR, an initial 2–5 minutes incubation is recommended to lyse cells
  • Avoid longer or higher temperature incubations (unless required due to high GC content of template)
  • Typically, a 15–30 second denaturation at 94°C should be utilized during thermocycling

Annealing Temperature and Duration

  • Match the primer Tms within 5°C of each other
  • Typical annealing temperatures are 5°C below the lowest primers Tm and often fall in the range of 45–60°C
  • Use the NEB Tm Calculator to calculate an appropriate annealing temperature.
  • Test higher annealing temperatures if spurious amplification products are observed
  • Typical annealing times are 15–60 seconds

Extension Time

  • Extensions are normally performed at 68°C
  • As a general rule, use extension times of one minute per 1000 base pairs (e.g., 3 minutes for a 3 kb product)
  • For products less than 1 kb, use 45–60 seconds
  • Products greater than 3 kb, or reactions using more than 30 cycles, may require longer extensions

Typical Cycling Conditions

Typical PCR protocol for a 500 bp amplicon

1 cycle 94°C 30 seconds
25 cycles 94°C 15 seconds
45-68°C 30 seconds
68°C 45 seconds
1 cycle 68°C 5 minutes (to finish replication on all templates)
1 cycle 4-10°C indefinite period (storing the sample prior to further analysis)


  1. Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA, 91, 2216-2220. PMID: 8134376


OneTaq® is a registered trademark of New England Biolabs, Inc.
Deep Vent® is a trademark of New England Biolabs, Inc.