Protocol for a PCR reaction using NEBNext® Q5® Hot Start HiFi PCR Master Mix (M0543)

PCR:
 
Please note that protocols with NEBNext Q5 Hot Start HiFi PCR Master Mix may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.

Reaction setup:

NEBNext Q5 Hot Start HiFi PCR Master Mix is inhibited at room temperature, allowing flexible reaction setup (RT or ice). 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 RNA
10 ng-1 μg
10 ng
NEBNext Q5
Hot Start HiFi PCR
Master Mix
25 μl 25 μl 25 μl 25 μl 25 μl
10 μM Primer 5 μl 2.5 μl 2.5 μl 2.5 μl 2.5 μl
10 μM Primer 5 μl 2.5 μl 2.5 μl 2.5 μl 2.5 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*
10 seconds
75 seconds
2-15 Cycles (depending on starting material)
Final Extension 65°C
5 minutes
1
Hold 4-10°C

 
*65°C is optimal for Illumina sample preparation.
Depending on primer design, annealing temperature may need to be optimized.

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:

    STARTING MATERIAL PRODUCT # AMOUNT CYCLES
    DNA E6000 and E6040 1 μg–5 μg 2–4
    E7370 5 ng–1 μg 4–12
    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 Q5 Hot Start HiFi 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. 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 Q5 Hot Start HiFi 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 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 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, 4–12 cycles yield sufficient product.

  9. PCR product: 

    The PCR products generated using NEBNext Q5 Hot Start HiFi PCR Master Mix have blunt ends.


  10. Bead Compatibility:

    The NEBNext Q5 Hot Start HiFi PCR Master Mix is compatible with a variety of carboxylated, tosylated and streptavidin beads that may be carried over or included in the PCR step of library construction protocols including 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.