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  • Guidelines for PCR Optimization with Thermophilic DNA Polymerases

    PCR for the purpose of cloning or other procedures requiring a low error rate benefits from thermophilic, proofreading DNA polymerases. These enzymes make far fewer mistakes during amplification and increase the odds of producing an error-free amplicon. New England Biolabs offers several proofreading DNA Polymerases including Q5™ (NEB #M0491), Phusion® (NEB #M0530), Q5™ Hot Start (NEB #M0493) Phusion® Hot Start Flex (NEB #M0535), OneTaq® (NEB #M0480), LongAmp® Taq (NEB #M0323), VentR® (NEB #M0254) and Deep VentR® (NEB #M0258) DNA polymerases. The increased accuracy of these polymerases stems from a 3´→ 5´ proofreading exonuclease. It is important to note that these polymerases can also degrade single stranded primers.

    Guidelines

    DNA Template

    • Use high quality, purified DNA templates. For colony PCR, use OneTaq DNA Polymerase
    • Approximately 104 copies of target DNA are required to detect product in 25-30 PCR cycles
    • Use 1pg–1ng of plasmid DNA
    • Use 10–100 ng of cosmid or cDNA template
    • 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 (i.e., 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 50-72°C. Click here to use our Tm calculator.
    • 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
    • When engineering sites into the end of primers, 4-6 extra bases should be added 5´ to the site
    • Final concentration should be 0.05-1.0 µM of each primer
    • Higher concentrations may increase secondary priming and create spurious amplification products
    • Primer degradation by the enzyme’s proofreading activity is inhibited by incorporating phosphorothioate linkages into the 2 bases at the 3´ end (1); however, this is usually not necessary

    Magnesium Concentration

    • 1.5–2.0 mM Mg2+ is typically optimal for OneTaq DNA Polymerase products.
    • 0.5–1.0 mM Mg2+ above the total dNTP concentration is optimal for Q5, Q5 Hot Start, Phusion and Phusion Hot Start Flex polymerases
    • If further optimization for Phusion is needed, increase the Mg2+ in 0.2 mM increments
    • 2.0 mM Mg2+ is typically optimal for VentR, Deep VentR and LongAmp Taq DNA Polymerases
    • VentR and Deep VentR may require Mg2+ titration in 2 mM increments up to 8 mM final concentration
    • Optimal concentration depends on template, buffer, DNA and dNTPs (each may chelate Mg2+)
    • If [Mg2+] is too low, no PCR product will be seen
    • If [Mg2+] is too high, undesired PCR products may be visible

    Deoxynucleotide Triphosphates (dNTPs)

    • Typical concentration is 200 µM of each dNTP
    • 300 µM of each dNTP is recommended for LongAmp Taq products

    High-Fidelity DNA Polymerase

    • The amount of DNA polymerase in the reaction can significantly affect PCR performance
    • Use 0.5–1 unit per 50 µl reaction of Q5, Phusion, Vent or Deep Vent Polymerase
    • 1–5 units per 50 µl reaction is recommended with LongAmp Taq products
    • Q5, Phusion, VentR and Deep VentR DNA Polymerases do not efficiently read through deoxyuridine or deoxyinosine in the template DNA strand, but OneTaq DNA Polymerase can.

    Denaturation Temperature and Duration

    • Initial denaturation at 94–98°C for 30 seconds 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 5–30 second denaturation should be utilized during thermocycling

    Annealing Temperature and Duration

    • Primer pairs should have Tms within 5°C of each other
    • Typical annealing temperatures are 0–2°C below the lowest primer Tm and often fall in the range of 50–60°C, for most polymerases other than Q5 and Phusion
    • Typical annealing temperatures are 0–3°C higher than the lowest Tm when using Q5 and Phusion
    • Tm s should be calculated using the nearest neighbor method when using Q5 or Phusion. Click here to use our Tm calculator.
    • Test higher annealing temperatures if extra amplification products are observed
    • Typical annealing times are 15–30 seconds

    Extension Time

    • Extensions are normally performed at 68–72°C; 65°C is optimal for LongAmp Taq Products
    • For Q5 and Phusion DNA Polymerases use extension times of 15 sec per 1000 base pairs for low complexity DNA and 30 sec per 1,000 base pairs for high complexity DNA templates
    • For OneTaq, Vent and Deep Vent DNA Polymerases use extension times of one minute per 1,000 base pairs (e.g., 3 minutes for a 3 kb product)
    • LongAmp Taq enzymes should be used at 50 seconds per 1,000 base pairs.
    • It is not advisable to use longer than recommended extension times when using proofreading polymerases

    Typical Cycling Conditions for OneTaq or OneTaq Hot Start DNA Polymerases

    Typical PCR protocol for a 1,000 bp amplicon

    1 cycle 94°C 30 seconds
    30 cycles 94°C 15 seconds
    55°C 15–30 seconds
    68°C 1 minute
    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)

    Typical Cycling Conditions for VentR or Deep VentR

    Typical PCR protocol for a 1,000 bp amplicon

    1 cycle 94°C 30 seconds
    25 cycles 94°C 15 seconds
    55°C 15 seconds
    72°C 1 minute
    1 cycle 72°C 5 minutes (to finish replication on all templates)
    1 cycle 4-10°C indefinite period (storing the sample prior to further analysis)

    Typical Cycling Conditions for Q5 or Phusion

    Typical PCR protocol for a 1,000 bp amplicon

    1 cycle 98°C 30 seconds
    25 cycles 98°C 10 seconds
    55°C 15 seconds
    72°C 15 seconds
    1 cycle 72°C 5 minutes (to finish replication on all templates)
    1 cycle 4-10°C indefinite period (storing the sample prior to further analysis)

    Typical PCR protocol for a 1,000 bp amplicon

    1 cycle 94°C 30 seconds
    30 cycles 94°C 10-30 seconds
    45-65°C 15-30 seconds
    65°C 50 seconds/kb
    1 cycle 65°C 10 minutes (to finish replication on all templates)
    1 cycle 4-10°C indefinite period (storing the sample prior to further analysis)

    Reference

    1. de Noronha, C. and Mullins, J. (1992) PCR Methods and Applications, 2, 131–136. PMID: 1335814

    Q5™ and Deep VentR™ are trademarks of New England Biolabs, Inc.
    OneTaq®, LangAmp Taq® and VentR® are registered trademarks of New England Biolabs.
    Phusion® was developed by Finnzymes Oy, now a part of Themo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. under agreement with, and under the performance specification of Themo Fisher Scientific.