HomeProtocolsPCR Using Q5® High-Fidelity DNA Polymerase (M0491)
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- Please note that protocols with Q5High-Fidelity DNA Polymerase may differ from protocols with other polymerases.Conditions recommended below should be used for optimalperformance.
Reaction Setup:
We recommendassembling all reaction components on ice and quickly transferring the reactionsto a thermocycler preheated to the denaturation temperature (98°C). Allcomponents should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may bediluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipettingerrors.
Notes: Gently mix thereaction. Collect all liquid to the bottom of the tube by a quick spin ifnecessary. Overlay the sample with mineral oil if using a PCR machine without aheated lid.COMPONENT 25 µl REACTION 50 µl REACTION FINAL CONCENTRATION 5X Q5
Reaction Buffer5 µl 10 µl 1X 10 mM dNTPs 0.5 µl 1 µl 200 µM 10 µM Forward Primer 1.25 µl 2.5 µl 0.5 µM 10 µM Reverse Primer 1.25 µl 2.5 µl 0.5 µM Template DNA variable variable < 1,000 ng Q5 High-Fidelity DNA Polymerase 0.25 µl 0.5 µl 0.02 U/µl 5X Q5 High GC Enhancer (optional) (5 µl) (10 µl) (1X) Nuclease-Free Water to 25 µl to 50 µl Transfer PCR tubes to a PCR machine and beginthermocycling.
Thermocycling Conditions for a RoutinePCR:
*Use of the NEBTmCalculator ishighly recommended.STEP TEMP TIME Initial Denaturation 98°C 30 seconds 25–35 Cycles 98°C
*50–72°C
72°C5–10 seconds
10–30 seconds
20–30 seconds/kbFinal Extension 72°C 2 minutes Hold 4–10°C - GeneralGuidelines:
Template:
Use of high quality, purified DNAtemplates greatly enhances the success of PCR. Recommended amounts of DNAtemplate for a 50 µl reaction are as follows:DNA AMOUNT DNA Genomic 1 ng–1 µg Plasmid or Viral 1 pg–10 ng - Primers:
Oligonucleotide primers aregenerally 20–40 nucleotides in length and ideally have a GC content of 40–60%.Computer programs such as Primer3 can be used to design or analyze primers. The bestresults are typically seen when using each primer at a final concentration of0.5 µM in the reaction. - Mg++ andadditives:
Mg++ concentration of 2.0 mM is optimal for most PCRproducts generated with Q5 High-Fidelity DNA Polymerase. When used at a finalconcentration of 1X, the Q5 Reaction Buffer provides the optimal Mg++concentration.Amplification of some difficult targets, like GC-richsequences, may be improved by the addition of 1X Q5 High GC Enhancer. The Q5High GC Enhancer is not a buffer and should not be used alone. It should beadded only to reactions with the Q5 Reaction Buffer when other conditions havefailed.
- Deoxynucleotides:
The finalconcentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended foruse with uracil-containing primers or templates. - Q5 High-Fidelity DNA Polymeraseconcentration:
We generally recommend using Q5 High-Fidelity DNA Polymeraseat a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, theoptimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40units/ml (0.5–2 units/50 μl reaction) depending on amplicon length anddifficulty. Do not exceed 2 units/50 μl reaction, especially for ampliconslonger than 5 kb. - Buffers:
The 5X Q5 Reaction Bufferprovided with the enzyme is recommended as the first-choice buffer for robust,high-fidelity amplification. For difficult amplicons, such as GC-rich templatesor those with secondary structure, the addition of the Q5 High GC Enhancer canimprove reaction performance. The 5X Q5 Reaction Buffer is detergent-free andcontains 2.0 mM Mg++at the final (1X)concentration. - Denaturation:
An initial denaturationof 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates.Longer denaturation times can be used (up to 3 minutes) for templates thatrequire it.During thermocycling, the denaturation step should be kept toa minimum. Typically, a 5–10 second denaturation at 98°C is recommended for mosttemplates.
- Annealing:
Optimal annealingtemperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than forother PCR polymerases. The NEB Tm Calculatorshould be used to determine the annealingtemperature when using this enzyme. Typically, use a 10–30 second annealing stepat 3°C above the Tm of the lower Tm primer. A temperaturegradient can also be used to optimize the annealing temperature for each primerpair.For high Tm primer pairs, two-step cycling without aseparate annealing step can be used (see note 12).
- Extension:
The recommended extensiontemperature is 72°C. Extension times are generally 20–30 seconds per kb forcomplex, genomic samples, but can be reduced to 10 seconds per kb for simpletemplates (plasmid, E. coli, etc.) or complex templates < 1 kb.Extension time can be increased to 40 seconds per kb for cDNA or long, complextemplates, if necessary.A final extension of 2 minutes at 72°C isrecommended.
- Cycle number:
Generally, 25–35 cyclesyield sufficient product. For genomic amplicons, 30-35 cycles are recommended. - 2-step PCR:
When primers with annealingtemperatures ≥ 72°C are used, a 2-step thermocycling protocol (combiningannealing and extension into one step) is possible. - Amplification of long products:
Whenamplifying products > 6 kb, it is often helpful to increase the extension time to 40–50seconds/kb. - PCR product:
The PCR products generatedusing Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the nextstep, then blunt-end cloning is recommended. If T/A-cloning is preferred, theDNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerasewill degrade any overhangs generated.Addition of an untemplated -dA canbe done with Taq DNA Polymerase (NEB#M0267 ) or Klenow exo– (NEB#M0212 ).
