• My NEB
  • Print
  • PDF
  • Guidelines for PCR Optimization with Taq DNA Polymerase

    Taq DNA Polymerase (NEB #M0273) is the enzyme most widely used in the Polymerase Chain Reaction (PCR). The following guidelines will help ensure the success of PCR using New England Biolabs’ Taq DNA Polymerase for routine PCR. Amplification of templates with high GC content, strong secondary structure, low concentrations or which produce products greater than 5 kb may require adaptation of these conditions.

    Learn about OneTaq® and OneTaq® Hot Start, NEB's new Taq-based polymerases for exceptional performance across a wide range of templates.

    Guidelines

    DNA Template

    • Use high quality, purified DNA templates
    • Approximately 104 copies of target DNA are required to detect product in 25-30 PCR cycles
    • Use 1pg–1ng of plasmid or viral templates
    • Use 1ng–1µg 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)

    Primers

    • Generally 20-30 nucleotides in length
    • Ideal GC content is 40-60%
    • Space GC residues evenly within the primer
    • Calculated melting temperatures (Tm) should be from 42-65°C
    • Use the NEB Tm calculator to determine the optimal annealing temperature 
    • 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 sites into the end of primers, 4-6 extra bases should be added 5´ to the site
    • Final concentration should be 0.05-1 µM, typically 0.1-0.5 µM of each primer
    • Higher concentrations may increase secondary priming and create spurious amplification products

    Magnesium Concentration

    • 1.5-2.0 mM is optimal for Taq 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 0.5 increments up to 4 mM

    Deoxynucleotides (dNTPs)

    • Typical concentration is 200 µM of each dNTP.
    • 50-100 µM enhances fidelity of polymerization, but reduces yields
    • Higher concentrations increase yields particularly in long PCR, but can reduce fidelity

    Taq DNA Polymerase Concentration

    • Use 0.5–2.0 units per 50 µl reaction, ideally 1.25 units

    Starting Reactions

    • Assemble all reaction components on ice
    • Add polymerase last
    • Immediately transfer reactions to thermocycler preheated to denaturation temperature (95°C)

    Denaturation Temperature and Duration

    • Initial denaturation at 95°C for 2 minutes is recommended prior to PCR cycling to fully denature the DNA
    • Avoid longer or higher temperature incubations (unless required due to high-GC content of template)
    • Typically, a 15-30 second denaturation at 95°C should be utilized during thermocycling

    Annealing Temperature and Duration

    • Match the Tms within 5°C of each other
    • Typical annealing temperatures are 5°C below the lowest primer's Tm and often fall in the range of 50-60°C
    • Test higher annealing temperatures if spurious amplification products are observed
    • Typical annealing times are 15-30 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 95°C 2 minutes
    25 cycles 95°C 15 seconds
    55°C 15 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)