Protocol for a PCR reaction using NEBNext® Ultra™ II Q5® Master Mix (M0544)



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.

Reaction setup:

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.

Component VOLUME
PER 50 μl RXN
NEBNext Ultra II
Q5 Master Mix
25 μl 1X
Primer pair variable 1 μM
Adaptor-ligated DNA* 15 μl variable
Nuclease-free water to 50 μl N/A
*From NEBNext DNA Library Prep Kit E6000, E6040, E7370 and E7645.

Transfer PCR tubes to a PCR machine and begin thermocycling.

Recommended Thermocycling Conditions:

Initial Denaturation
30 seconds


10 seconds

75 seconds

2-15 Cycles
on starting
Final Extension 65°C
5 minutes
Hold 4-10°C

* 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.

General Guidelines: 

  1. 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 follows:
    E6000 & E6040 1 μg–5 μg 2–4
    E7370 5 ng–1 μg 4–12
    E7645 0.5 ng–1 μg 3–15

    * Please refer to the specific product manual for detailed recommended cycle numbers tailored to your DNA input.

  2. 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.

  3. Deoxynucleotides:

    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.

  4. 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.

  5. Denaturation:

    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 templates.

  6. Annealing:

    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.

  7. Extension:

    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 time.

    A final extension of 5 minutes at 65°C is recommended.

  8. 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 recommendations

  9. 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.