PCR Optimization with Phusion® High-Fidelity PCR Kit

Introduction

The following guidelines are provided to ensure successful PCR using Phusion DNA Polymerase. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations or longer amplicons may require further optimization.

Guidelines

1. Template:
   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 using Phusion DNA Polymerase may be 0.2–1 μM, while 0.5 μM is recommended.
   
   
3. Mg⁺⁺ and additives:
   Mg⁺⁺ is critical to achieve optimal activity with Phusion DNA Polymerase. The final Mg⁺⁺ concentration in 1X Phusion HF and GC Buffer is 1.5 mM. Excessive Mg⁺⁺ can prevent full denaturation of DNA as well as cause non-specific binding of primers. The optimal Mg⁺⁺ concentration is affected by dNTP concentration, the template being used and supplements that are added to the reaction. This can also be affected by the presence of chelators (e.g. EDTA). Mg⁺⁺ can be optimized in 0.5 mM increments using the MgCl₂  provided.
   
   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 it decreases the primer T_m. Phusion DNA polymerase is also compatible with other additives such as formamide or glycerol. 
   
   
4. Deoxynucleotides:
   The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Phusion cannot incorporate dUTP.
   
   
5. Phusion DNA Polymerase Concentration:
   We generally recommend using Phusion DNA Polymerase at a concentration of 20 units/ml (1.0 units/50 μl reaction). However, the optimal concentration of Phusion DNA Polymerase may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 µl reaction, especially for amplicons longer than 5 kb.
   
   
6. Buffers:
   5X Phusion HF Buffer and 5X Phusion GC Buffer are provided with the enzyme. HF buffer is recommended as the default buffer for high-fidelity amplification. For difficult templates, such as GC-rich templates or those with secondary structure, GC buffer can improve reaction performance. GC buffer should be used in experiments where HF buffer does not work. Detergent-free reaction buffers are also available for applications that do not tolerate detergents (e.g. microarray, DHPLC).
   
   
7. Denaturation:
   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. 
   
   
8. Annealing:
   Annealing temperatures required for use with Phusion tend to be higher than with other PCR polymerases. The NEB T_m Calculator should be used to determine the annealing temperature when using Phusion. Typically, primers greater than 20 nucleotides in length anneal for 10–30 seconds at 3°C above the T_m of the lower T_m primer. If the primer length is less than 20 nucleotides, an annealing temperature equivalent to the T_m 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 T_m primer pairs, two-step cycling without a separate annealing step can be used.
   
   
9. 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. 
   
   
10. Cycle number:
    Generally, 25–35 cycles yields sufficient product. 
    
    
11. 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:

    Cycle step	Temp	Time
    Initial denaturation	98°C	30 seconds
    25–35 Cycles	98°C 72°C	5–10 seconds 15–30 seconds per kb
    Final extension	72°C	5–10 minutes
    Hold	4°C	∞

    
    
    
12. PCR product:
    The PCR products generated using Phusion DNA Polymerase have blunt ends; if cloning is the next step, then blunt-end cloning is recommended. If TA-cloning is preferred, then DNA should be purified prior to A-addition, as Phusion 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).