Phusion Hot Start DNA Polymerase Guidelines for PCR Reactions
The optimal amount of enzyme depends on the amount of template and the length of the PCR product. Usually 1 unit of Phusion Hot Start DNA Polymerase per 50 µl reaction volume gives good results, but optimal amounts could range from 0.5-2 units per 50 µl reaction depending on amplicon length and difficulty. Do not exceed 2 U/50 µl (0.04 U/µl), especially for amplicons that are > 5kb.
When cloning fragments amplified with Phusion Hot Start DNA Polymerase, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product with e.g. Taq DNA Polymerase (NEB #M0267S/L or #M0273S/L). However, before adding the overhangs it is very important to remove all the Phusion Hot Start DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion Hot Start DNA Polymerase is very strong at 72°C. Any remaining Phusion Hot Start DNA Polymerase will degrade the A overhangs, thus creating the blunt ends again. A detailed protocol for TA cloning of Phusion PCR products can be found on Finnzymes' website www.finnzymes.com.
Two buffers are provided with the enzyme: 5X Phusion HF Buffer and 5X Phusion GC Buffer. The error rate of Phusion Hot Start DNA Polymerase in HF Buffer (4.4 x 10-7) is lower than that in GC Buffer (9.5 x 10-7). Therefore, the HF Buffer should be used as the default buffer for high-fidelity amplification. However, GC Buffer can improve the performance of Phusion Hot Start DNA Polymerase on some difficult or long templates, i.e. GC-rich templates or those with complex secondary structures. Use of GC Buffer is recommended when amplification with HF Buffer has failed.
- Mg2+ concentration and dNTP concentration
The concentration of Mg2+ is critical since Phusion Hot Start DNA Polymerase is a magnesium-dependent enzyme. Excessive Mg2+ stabilizes the DNA double strand and prevents complete denaturation of DNA. Excess Mg2+ can also stabilize spurious annealing of primer to incorrect template sites and decrease specificity. Conversely, inadequate Mg2+ could lead to lower product yield. The optimal Mg2+ concentration depends on the dNTP concentration, the specific template DNA and the sample buffer composition. In general, the optimal Mg2+ concentration is from 0.5 to 1 mM over the total dNTP concentration for standard PCR. If the primers and/or template contain chelators such as EDTA or EGTA, the apparent Mg2+ optimum may be shifted to higher concentrations. If further optimization is needed, increase Mg2+ concentration in 0.2 mM steps.
High quality dNTPs (e.g. F-560S/L) should be used for optimal performance with Phusion Hot Start DNA Polymerase. Use of dUTP and other dUTP-derivatives or analogues is not recommended. Due to the high processivity of Phusion Hot Start DNA Polymerase there is no advantage of increasing dNTP concentrations. For optimal results always use 200 µM of each dNTP.
General guidelines are: 1 pg - 10 ng / 50 µl reaction with low complexity DNA (e.g. plasmid, lambda or BAC DNA); 50-500 ng / 50 µl reaction with high complexity genomic DNA.
- PCR additives
The recommended reaction conditions for GC -rich templates include 3% DMSO as a PCR additive, which aids in the denaturing of templates with high GC contents. For further optimization DMSO should be varied in 2% increments. In some cases DMSO may also be required for supercoiled plasmids to relax for denaturation. Other PCR additives such as formamide (up to 3%), glycerol, and betaine are also compatible with Phusion Hot Start DNA Polymerase.
If high DMSO concentration is used, the annealing temperature must be lowered, as DMSO decreases the melting point of the primers. It has been reported that 10% DMSO decreases the annealing temperature by 5.5-6.0°C.
- Initial denaturation
Denaturation should be done at 98°C (calculated sample temperature). Due to the high thermostability of Phusion Hot Start DNA Polymerase even higher than 98°C denaturation temperatures can be used. We recommend 30 s initial denaturation at 98°C for most templates. Some templates may require longer initial denaturation and the length of the initial denaturation time can be extended up to 3 minutes.
Keep the denaturation as short as possible. Usually 5-10 seconds at 98°C is enough for most templates. Note: the denaturation time and temperature may vary depending on the ramp rate and temperature control mode of the cycler.
- Primer annealing
Phusion Hot Start DNA Polymerase has the ability to stabilize primer-template hybridization. As a basic rule, for primers >20nt, anneal for 10-30 seconds at a Tm +3°C of the lower Tm primer. The Tm's should be calculated with the nearest-neighbor method because results from primer Tm calculations can vary significantly depending on the method used. For primers <= 20nt, use an annealing temperature equal to the Tm of the lower Tm primer. If necessary, use a temperature gradient to find the optimal annealing temperature for each template-primer pair combination. The annealing gradient should extend up to the extension temperature (two-step PCR). Two-step cycling without an annealing step is also recommended for high Tm primer pairs. Instructions for Tm calculation and a link to a calculator using the nearest-neighbor method can be found on Finnzymes' website (www.finnzymes.com).
The extension should be performed at 72°C. The extension time depends on the length and complexity of the amplicon. For low complexity DNA (e.g. plasmid, lambda or BAC DNA) use extension time 15 s per 1kb. For high complexity genomic DNA, 30 s per 1kb is recommended.