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- Please note that protocols with Q5
High-Fidelity DNA Polymerase may differ from protocols with other polymerases.
Conditions recommended below should be used for optimal
assembling all reaction components on ice and quickly transferring the reactions
to a thermocycler preheated to the denaturation temperature (98°C). All
components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be
diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting
Notes: Gently mix the
reaction. Collect all liquid to the bottom of the tube by a quick spin if
necessary. Overlay the sample with mineral oil if using a PCR machine without a
||25 µl REACTION
||50 µl REACTION
|10 mM dNTPs
|10 µM Forward Primer
|10 µM Reverse Primer
||< 1,000 ng
|Q5 High-Fidelity DNA Polymerase
|5X Q5 High GC Enhancer (optional)
||to 25 µl
||to 50 µl
Transfer PCR tubes to a PCR machine and begin
Thermocycling Conditions for a Routine
*Use of the NEB Tm Calculator is
Use of high quality, purified DNA
templates greatly enhances the success of PCR. Recommended amounts of DNA
template for a 50 µl reaction are as follows:
||1 ng–1 µg
|Plasmid or Viral
||1 pg–1 ng
Oligonucleotide primers are
generally 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 best
results are typically seen when using each primer at a final concentration of
0.5 µM in the reaction.
- Mg++ and
Mg++ concentration of 2.0 mM is optimal for most PCR
products generated with Q5 High-Fidelity DNA Polymerase. When used at a final
concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg++
Amplification of some difficult targets, like GC-rich
sequences, may be improved by the addition of 1X Q5 High GC Enhancer. The Q5
High GC Enhancer is not a buffer and should not be used alone. It should be
added only to reactions with the Q5 Reaction Buffer when other conditions have
concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5
High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for
use with uracil-containing primers or templates.
- Q5 High-Fidelity DNA Polymerase
We generally recommend using Q5 High-Fidelity DNA Polymerase
at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the
optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40
units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and
difficulty. Do not exceed 2 units/50 μl reaction, especially for amplicons
longer than 5 kb.
The 5X Q5 Reaction Buffer
provided with the enzyme is recommended as the first-choice buffer for robust,
high-fidelity amplification. For difficult amplicons, such as GC-rich templates
or those with secondary structure, the addition of the Q5 High GC Enhancer can
improve reaction performance. The 5X Q5 Reaction Buffer is detergent-free and
contains 2.0 mM Mg++ at the final (1X)
An initial denaturation
of 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 that
During thermocycling, the denaturation step should be kept to
a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most
temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for
other PCR polymerases. The NEB
Tm Calculator should be used to determine the annealing
temperature when using this enzyme. Typically, use a 10–30 second annealing step
at 3°C above the Tm of the lower Tm primer. A temperature
gradient can also be used to optimize the annealing temperature for each primer
For high Tm primer pairs, two-step cycling without a
separate annealing step can be used (see note 12).
The recommended extension
temperature is 72°C. Extension times are generally 20–30 seconds per kb for
complex, genomic samples, but can be reduced to 10 seconds per kb for simple
templates (plasmid, E. coli, etc.) or complex templates < 1 kb.
Extension time can be increased to 40 seconds per kb for cDNA or long, complex
templates, if necessary.
A final extension of 2 minutes at 72°C is
- Cycle number:
Generally, 25–35 cycles
yield sufficient product. For genomic amplicons, 30-35 cycles are recommended.
- 2-step PCR:
When primers with annealing
temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining
annealing and extension into one step) is possible.
- Amplification of long products:
amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50
- PCR product:
The PCR products generated
using Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the next
step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the
DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerase
will degrade any overhangs generated.
Addition of an untemplated -dA can
be done with Taq DNA Polymerase (NEB
#M0267 ) or Klenow exo– (NEB