PCR Using NEBNext® High-Fidelity 2X PCR Master Mix (M0541)

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 performance.

Reaction Setup:
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.

Component DNA
PROTOCOL
mRNA PROTOCOL ChIP DNA
PROTOCOL
1 μg-5 μg 5 ng-1 μg 50 ng-250 ng
PURIFIED mRNA
TOTAL mRNA
10 ng-1 ng
10 ng
NEBNext High-Fidelity
2X PCR Master Mix
25 µl 25 µl 25 µl 25 µl 25 µl
25 μM Primer 2.5 µl 1 µl 1 µl 1 µl 1 µl
25 μM Primer 2.5 µl 1 µl 1 µl 1 µl 1 µl
Adaptor-ligated DNA 20 µl 23 µl 23 µl 20 µl 23 µl
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: 
STEP TEMP TIME CYCLES
Initial Denaturation 98°C 30 seconds 1
Denaturation
Annealing
Extension
98°C
65°C*
72°C
10 seconds
30 seconds
30 seconds
4-15 Cycles
(depending
on starting
material)
Final Extension 72°C 5 minutes 1
Hold 4°C    
*65°C is optimal for Illumina sample preparation. Depending on primer design, annealing temperature may need to be optimized. 

General Guidelines: 
  1. Template:
    Use of high quality, purified DNA templates greatly enhances the success of PCR.
    STARTING MATERIAL NEB# AMOUNT CYCLES
    DNA E6000 and
    E6040
    1 μg–5 μg 4–8
    E7370 5 ng–1 μg 6–15
    Purified mRNA E6100 and
    E6110
    50–250 ng 10–12
    Total RNA E7420 100 ng–1 μg 12–15
    E7530 10 ng–1 μg 12–15
    ChIP DNA E6200 and
    E6240
    10 ng 15
  2. Mg++ and additives:
    The 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.

  3. Deoxynucleotides:
    The final 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 templates.

  4. DNA polymerase concentration:
    The concentration of DNA Polymerase in the NEBNext High-Fidelity 2X PCR 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 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.

  7. Extension:
    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.

    A final extension of 5 minutes at 72°C is recommended.

  8. Cycle number:
    Generally, 4–15 cycles yield sufficient product.

  9. PCR product:
    The PCR products generated using NEBNext High-Fidelity 2X PCR Master Mix have blunt ends.

  10. Perform clean up after the PCR reaction using SPRI beads or PCR purification columns.