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. In addition, the protocol for NGS library prep ampliﬁcation and the protocol for standard end point PCR will diﬀer. Conditions recommended below should be used for optimal performance in either application.
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.
NGS PCR for use with NEBNext NGS oligos for Illumina
|Component||Volume per 50 µl RXN||Final Concentration|
|NEBNext Ultra II Q5 Master Mix||25 µl||1x|
|10 uM Forward Primer||5 µl*||1 µM|
|10 uM Reverse Primer||5 µl*||1 µM|
|Adaptor‐ligated DNA**||Variable||Variable (< 1 µg for E7645)|
*For NEBNext Index Primers as supplied in tubes (10 μM) use 5 μl from each primer
*For NEBNext Primers as supplied in 96 well plates (10 µM) use 10 µl of the combined primer
** Adaptor Ligated DNA made with NEBNext Ultra II DNA Library Prep for Illumina E7645
End Point PCR
|Component||Volume per 50 ul RXN||Final Concentration|
|NEBNext Ultra II Q5 Master Mix||25 µl||1x|
|10 uM Forward Primer||2.5 µl||0.5 µM|
|10 uM Reverse Primer||2.5 µl||0.5 µM|
|Template DNA||Variable||Variable (< 1000 ng for End Point PCR)|
Transfer PCR tubes to a PCR machine and begin thermocycling.
Recommended Thermocycling Conditions:
|Initial Denaturation||98°C||30 seconds||1|
|Annealing/ Extension*||65°C||75 seconds|
|Final Extension||65°C||5 minutes||1|
*For NGS primers other than NEBNext index primers the Tm may be diﬀerent. In that case use 30 Sec at Tm (use NEB Tm calculator) and 45 Sec at 65oC
**The number of cycles depends on the input amount. Check the E7645 manual for a guideline for PCR cycle numbers. Further optimization to avoid over ampliﬁcation may be required.
|Initial Denaturation||98oC||30 seconds||1|
|Extension||72°C||20‐30 seconds/ kb|
|Final Extension||72°C||2 minutes||1|
***Use of the NEB Tm Calculator is highly recommended. Further optimization may be required.
For NGS PCR
Use of high quality, puriﬁed DNA templates greatly enhances the success of library ampliﬁcation reactions. Recommended amounts of DNA template for a 50 μl reaction are between 0.5 ng - 1 µg as per the E7645 Product Manual.
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 templates.
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. For NEBNext multiplex PCR primers the annealing temperature is 65°C. If a diﬀerent annealing temperature is used, anneal at that temperature for 30 seconds.
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.
Generally, 3 - 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
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.
For Standard PCR
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:
DNA AMOUNT DNA Genomic 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. Q5 is a high fidelity polymerase and for NGS amplification we would recommend using a primer with a 3' phosphorothioate modification.
Mg++ and additives:
The Q5 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 mix.
The ﬁnal concentration of dNTPs is optimized for robust library ampliﬁcation. Q5 DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil‐containing primers or templates.
NEBNext Ultra II Q5 Master Mix DNA Polymerase concentration:
The concentration of Q5 DNA Polymerase in the NEBNext Ultra II Q5 Master Mix has been optimized for best results under a wide range of conditions.
NEBNext Ultra II Q5 Hot Master Mix does not require a separate activation step.
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.
Optimal annealing temperatures for NEBNext Ultra II Q5 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 pair. For high Tm primer pairs, two‐step cycling without a separate annealing step can be used (see note 1).
The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic sam‐ ples, 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 recommended.
Generally, 25 - 35 cycles yield suﬃcient product. For genomic amplicons, 30‐35 cycles are recommended.
When primers with annealing temperatures ≥ 72°C are used, a 2‐step thermocycling protocol (combining annealing and extension into one step) is possible.
The PCR products generated using NEBNext Ultra II Q5 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 generated. Addition of an untemplated -dA can be done with Taq DNA Polymerase (NEB #M0267) or Klenow exo– (NEB #M0212).