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  • Protocol for Phusion® Hot Start Flex 2X Master Mix


    The following guidelines are provided to ensure successful PCR using Phusion® Hot Start Flex 2X Master Mix. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations or long amplicons may require further optimization.


    1. Reaction Setup: Phusion Hot Start Flex DNA Polymerase is inhibited at room temperature, allowing flexible reaction set up (RT or ice). All components should be mixed and spun down prior to use. Please note that protocols with Phusion Hot Start Flex DNA Polymerase may differ from protocols with other standard polymerases. As such, conditions recommended below should be used for optimal performance.

      Nuclease-free water to 25 µl to 50 µl  
      10 μM Forward Primer 1.25 µl 2.5 µl 0.5 µM
      10 μM Reverse Primer 1.25 µl 2.5 µl 0.5 µM
      Template DNA variable variable < 250 ng
      DMSO (optional) (0.75 µl) (1.5 µl) 3%
      Phusion Hot Start Flex 2X Master Mix 12.5 µl 25 µl 1X
      Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

      Transfer PCR tubes to a PCR machine and begin thermocycling. Phusion Hot Start Flex DNA Polymerase does not require a separate activation step. Standard Phusion cycling conditions are recommended.
      Thermocycling conditions for a routine PCR:
      Initial denaturation:
      98°C   30 seconds

      25–35 cycles:
      98°C   5–10 seconds
      45–72°C*   10–30 seconds
      72°C   15–30 seconds/kb 

      Final extension:
      72°C   5–10 minutes


      *To determine the optimal annealing temperature for a given set of primers, use of the NEB Tm Calculator   is highly recommended

      General Guidelines:

      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
      Genomic 50 ng–250 ng
      Plasmid or viral 1 pg–10 ng

      If the template DNA is obtained from a cDNA synthesis reaction, the volume added should be less than 10% of the total reaction volume. 

    2. Primers:
      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 final concentration of each primer in a PCR experiment using Phusion Hot Start Flex DNA Polymerase may be 0.2–1 μM, while 0.5 μM is strongly recommended.

    3. Mg++ and additives:
      At 1X concentration, Phusion Hot Start Flex Master Mix provides 1.5 mM MgCl2 and 200 μM of each dNTP in the final reaction. Phusion Hot Start Flex DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or template.

      Amplification of difficult targets, such as those with GC-rich sequences or secondary structure, may be improved by the presence of additives such as DMSO (included). A final concentration of 3% DMSO is recommended, although concentration can be optimized in 2% increments. It is important to note that if a high concentration of DMSO is used, the annealing temperature must be lowered as DMSO decreases the primer Tm (2). Phusion Hot Start Flex 2X Master Mix is also compatible with other additives such as formamide or glycerol.

    4. Phusion Hot Start Flex DNA Polymerase Concentration:
      The concentration of Phusion Hot Start Flex DNA Polymerase in the Phusion Hot Start Flex 2X Master Mix has been optimized for best results under a wide range of conditions. If reactions are set up according to recommendations listed, the final concentration of Phusion Hot Start Flex DNA Polymerase is 1 unit/50 μl reaction or 0.5 units/25 μl reaction.

    5. Denaturation:
      Phusion Hot Start Flex DNA Polymerase 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.

    6. Annealing:
      Optimal annealing temperatures for Phusion Hot Start Flex DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator   should be used to determine the annealing temperature when using Phusion products. Typically, for primers > 20 nt, use a 10–30s annealing step at 3°C above the Tm of the lower Tm primer. For primers < 20 nt, an annealing temperature equivalent to the Tm of the lower primer should be used. A temperature gradient can also be used to optimize the annealing temperature for each primer pair. For two-step cycling, the gradient can be set as high as the extension temperature.

      For high Tm primer pairs, two-step cycling without a separate annealing step can be used.

    7. Extension:
      The recommended extension temperature is 72°C. Extension times are dependent on amplicon length and complexity. Generally, an extension time of 15 seconds per kb can be used. For complex amplicons, such as genomic DNA, an extension time of 30 seconds per kb is recommended. Extension time can be increased to 40 seconds per kb for cDNA templates, if necessary.

    8. Cycle number:
      Generally, 25–35 cycles yield sufficient product.

    9. 2-step PCR:
      When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol is recommended. 

      Thermocycling Conditions for a Routine 2-Step PCR:

      Initial denaturation:
      98°C 30 seconds

      25–35 cycles:
      98°C 5–10 seconds
      72°C 15–30 seconds/kb

      Final extension:
      72°C 5–10 minutes


    10. PCR product:
      The PCR products generated using Phusion Hot Start Flex DNA Polymerase 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 Phusion Hot Start Flex 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).