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Please note that protocols with Q5® Hot Start High- Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
Q5 Hot Start High-Fidelity DNA Polymerase is inhibited at room temperature, allowing flexible reaction setup (RT or ice).
All components should be mixed prior to use. Q5 Hot Start High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.
||25 µl Reaction
||50 µl Reaction
|5X Q5 Reaction Buffer
|10 mM dNTPs
|10 µM Forward Primer
|10 µM Reverse Primer
||< 1,000 ng
|Q5 Hot Start High-Fidelity DNA Polymerase
|5X Q5 High GC Enhancer (optional)
||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.
Q5 Hot Start High-Fidelity DNA Polymerase does not require a separate activation step. Standard Q5 cycling conditions are recommended.
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 µ l 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 Hot Start High-Fidelity DNA Polymerase. When used at
a final concentration of 1X, the Q5 Reaction Buffer provides the optimal
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 failed.
concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5 Hot Start
High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for
use with uracil-containing primers or templates.
- Q5 Hot Start High-Fidelity DNA Polymerase
We generally recommend using Q5 Hot Start High-Fidelity DNA
Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction).
However, the optimal concentration of Q5 Hot Start 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 contains 2.0 mM
Mg++ at the final (1X) concentration.
Q5 Hot Start
High-Fidelity DNA Polymerase does not require a separate activation
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 require it. During thermocycling, the
denaturation step should be kept to a minimum. Typically, a 5–10 second
denaturation at 98°C is recommended for most templates.
temperatures for Q5 Hot Start 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 seconds 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 pair.
Tm primer pairs, two-step cycling without a separate annealing step
can be used (see note 11).
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 Hot Start 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 Hot Start
High-Fidelity DNA Polymerase will degrade any overhangs
Addition of an untemplated -dA can be done with Taq DNA Polymerase (NEB #M0267) or Klenow
exo– (NEB #M0212).