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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 performance.
We recommend 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.
||25 µl Reaction
||50 µl Reaction
|Q5 High-Fidelity 2X Master Mix
|10 µM Forward Primer
|10 µM Reverse Primer
||< 1,000 ng
||to 25 µl
||to 50 µl
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 heated lid.
Transfer PCR tubes to a PCR machine and begin thermocycling.
Thermocycling Conditions for a Routine PCR:
*Use of the NEB Tm Calculator
is highly recommended.
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 additives:
High-Fidelity Master Mix contains 2.0 mM Mg++ when used at a 1X
concentration. This is optimal for most PCR products generated with this master
concentration of dNTPs is 200 μM of each deoxynucleotide in the 1X Q5
High-Fidelity Master Mix. Q5 High-Fidelity DNA Polymerase cannot incorporate
dUTP and is not recommended for use with uracil-containing primers or
- Q5 High-Fidelity DNA Polymerase
The concentration of Q5 High-Fidelity DNA Polymerase in the Q5
High-Fidelity 2X Master Mix has been optimized for best results under a wide
range of conditions.
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 10).
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 seconds/kb.
- PCR product:
The PCR products generated
using Q5 High-Fidelity 2X Master Mix 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