Please note that protocols with NEBNext® High- Fidelity 2X
PCR Master Mix may differ from protocols with other polymerases. Conditions
recommended below should be used for optimal
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.
|1 μg-5 μg
||5 ng-1 μg
||50 ng-250 ng
10 ng-1 ng
2X PCR Master Mix
|25 μM Primer
|25 μM Primer
Notes: Gently mix the reaction. Collect all liquid at the bottom of the tube by a quick spin if necessary.
Transfer PCR tubes to a PCR machine and begin thermocycling.
Thermocycling conditions for a routine PCR:
*65°C is optimal for Illumina sample preparation. Depending on primer design, annealing temperature may need to be optimized.
Use of high quality, purified DNA templates greatly enhances the
success of PCR.
|1 μg–5 μg
||5 ng–1 μg
||100 ng–1 μg
||10 ng–1 μg
- Mg++ and additives:
NEBNext High-Fidelity 2X PCR 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.
concentration of dNTPs is 200 μM of each deoxynucleotide in the NEBNext High-
Fidelity 2X PCR Master Mix. Q5 High-Fidelity DNA Polymerase cannot incorporate
dUTP and is not recommended for use with uracilcontaining primers or
- DNA polymerase concentration:
concentration of DNA Polymerase in the NEBNext High-Fidelity 2X PCR Master Mix
has been optimized for best results under a wide range of
An initial denaturation
of 30 seconds at 98°C is sufficient for most sample types.
thermocycling, the denaturation step should be kept to a minimum. Typically, a
10 second denaturation at 98°C is recommended for most templates.
temperatures for NEBNext High Fidelity 2X PCR Master Mix 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 is 72°C. Extension times are generally 30 seconds for libraries up
to 1kb. Larger insert lengths may require additional time.
extension of 5 minutes at 72°C is recommended.
- Cycle number:
Generally, 4–15 cycles
yield sufficient product.
- PCR product:
The PCR products generated
using NEBNext High-Fidelity 2X PCR Master Mix have blunt ends.
- Perform clean up after the PCR reaction
using SPRI beads or PCR purification columns.