Please note that protocols with Q5
Hot Start High-Fidelity 2X Master Mix may differ from protocols with other
polymerases. Conditions recommended below should be used for optimal
Q5 Hot Start High-Fidelity 2X Master Mix is inhibited at room temperature, allowing flexible reaction setup (RT or ice).
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.
Transfer PCR tubes to a PCR machine and begin thermocycling.
Q5 Hot Start High-Fidelity 2X Master Mix 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 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 μ M of each deoxynucleotide in the 1X Q5 Hot Start
High-Fidelity Master Mix. Q5 Hot Start High-Fidelity DNA Polymerase cannot
incorporate dUTP and is not recommended for use with uracil-containing primers
- Q5 Hot Start High-Fidelity DNA Polymerase
The concentration of Q5 Hot Start High- Fidelity DNA
Polymerase in the Q5 Hot Start High-Fidelity 2X Master Mix has been optimized
for best results under a wide range of conditions.
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.
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 Hot Start High-Fidelity 2X Master Mix 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 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 seconds/kb.
- PCR product:
The PCR products generated
using Q5 Hot Start 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 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