Please note that protocols with NEBNext Ultra II Q5
Master Mix may differ from protocols with other
polymerases. Conditions recommended below
should be used for optimal performance.
NEBNext Ultra II Q5 Master Mix is inhibited at room
temperature, allowing flexible reaction setup (RT or
ice). All components should be mixed prior to use.
*From NEBNext DNA Library Prep Kit E6000, E6040, E7370 and E7645.
PER 50 μl RXN
|NEBNext Ultra II
Q5 Master Mix
||to 50 μl
Transfer PCR tubes to a PCR machine and begin thermocycling.
Recommended Thermocycling Conditions:
* The PCR conditions have been optimized for NEBNext Adaptor-ligated DNAs
with NEBNext primer sets. For user-supplied adaptors and primers, a threestep
PCR with a 45 second extension may be necessary depending on the
optimal annealing temperature for your primer.
Use of high quality, purified DNA templates greatly
enhances the success of PCR reactions. Recommended
amounts of DNA template for a 50 μl reaction are as
|E6000 & E6040
||1 μg–5 μg
||5 ng–1 μg
||0.5 ng–1 μg
* Please refer to the specific product manual for detailed recommended cycle numbers tailored to your DNA input.
- Mg++ and additives:
The NEBNext Ultra II Q5 Master Mix contains 2.0 mM Mg++ when used at a 1X concentration. This is optimal for most PCR products generated with this master mix.
The final concentration of dNTPs is optimized for robust
library amplification. Q5 High-Fidelity DNA Polymerase
cannot incorporate dUTP and is not recommended for
use with uracil-containing primers or templates.
- DNA polymerase concentration:
The concentration of DNA Polymerase in the NEBNext
Ultra II Q5 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 sample types.
During thermocycling, the denaturation
step should be kept to a minimum. Typically, a 10
second denaturation at 98°C is recommended for most
Optimal annealing temperatures for Q5 High-
Fidelity DNA Polymerase tend to be higher than
for other PCR polymerases. Depending on
primer design, the annealing temperature may
need to be optimized.
The recommended extension temperature for
library amplification is 65°C. Extension times
are generally 30 seconds for libraries up to
1 kb. Larger insert lengths may require additional
A final extension of 5 minutes at 65°C
- Cycle number:
Generally, 2–13 cycles yield sufficient product
depending on the DNA input and specific DNA
library prep product utilized. Please refer to
the product manual for detailed cycle number
- Bead Compatibility:
The NEBNext Ultra II Q5 Master Mix is
compatible with a variety of carboxylated and
tosylated beads that may be carried over or
included in the PCR step of library construction
protocols including streptavidin beads,
Agencourt® AMPure® XP (Beckman Coulter,
Inc.), Sera-Mag SpeedBeads and Mag-Bind®
RXNPure Plus (Omega Bio-tek, Inc.). SPRI Beads
or PCR purification columns are recommended
for post PCR clean up.