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Last updated
Last checked against the vendor documents listed under Sources on 2026-10-29. Research use only: the vendor documents state that these products are not for diagnostic procedures.
Short answer: the Phusion PCR protocol in one paragraph
A Phusion PCR protocol uses 0.5 µM of each primer, 200 µM of each dNTP and a 98 °C denaturation step, then 25-35 cycles with an extension time of 15-30 seconds per kilobase at 72 °C, never more than 1 minute per kilobase. Thermo Scientific states that the best annealing temperature for Phusion DNA Polymerase may differ significantly from the one used with Taq-based polymerases, so a protocol for PCR written for Taq does not transfer unchanged. The rest of this page gives the reaction table, the cycling table, a product chooser and a symptom-to-fix map, all taken from two Thermo Scientific documents.
This page covers the general PCR protocol for Phusion-family polymerases, also searched as “pcr phusion protocol” or “pcr protocol phusion”. For a vendor-neutral walk-through of reaction setup, see PCR protocol basics: how to set up a reaction.
What you need
Reagents
There are two ways to build the reaction, and the choice changes what you pipette.
- Phusion High-Fidelity PCR Master Mix (2X). The mix already contains the polymerase, nucleotides and a buffer with MgCl2. The vendor states that only template and primers need to be added by the user. The final concentrations are 1.5 mM MgCl2 and 200 µM of each dNTP. See master mix in PCR for what a master mix does and does not fix.
- Separate components. 5X Phusion buffer (HF or GC), 10 mM dNTPs, the polymerase, primers, template and water. Enzyme amount is 0.5-1.0 U per 50 µL, and the vendor states not to exceed 2 U per 50 µL.
Also needed: forward and reverse primers (see how to choose primers for PCR), template DNA (see PCR template DNA) and, optionally, DMSO.
Reaction table
| Component | 50 µL reaction | 20 µL reaction | Final concentration |
|---|---|---|---|
| Nuclease-free water | to 50 µL | to 20 µL | – |
| 2X Phusion Master Mix (master-mix route) | 25 µL | 10 µL | 1X |
| 5X Phusion HF or GC buffer (separate-component route) | 10 µL | 4 µL | 1X |
| 10 mM dNTPs (separate-component route only) | 1 µL | 0.4 µL | 200 µM each |
| Forward primer | – | – | 0.5 µM (range 0.2-1.0 µM) |
| Reverse primer | – | – | 0.5 µM (range 0.2-1.0 µM) |
| Template DNA | see below | see below | – |
| DMSO (optional) | 1.5 µL | 0.6 µL | 3% |
| Phusion polymerase (separate-component route only) | 0.5 µL | 0.2 µL | 0.02 U/µL |
Primer volumes depend on your stock concentration, so they are given as final concentrations. The master-mix route adds no dNTPs, because they are already in the 2X mix. Per 50 µL, the vendor gives these template amounts: 1 pg-10 ng of low-complexity DNA (plasmid, lambda, BAC) and 50-250 ng of genomic DNA. A cDNA reaction mix should be no more than 10% of the final volume.
Steps
- Set up on ice and add components in the order of the table: water first, then master mix (or buffer, dNTPs), primers, template, optional DMSO, and the polymerase last if you are using separate components.
- Choose the buffer. HF buffer is the default. The vendor recommends GC buffer for GC-rich, difficult or long templates.
- Mix gently, close the tubes and place them in the cycler.
- Run the cycling program below.
- Hold at 4 °C and analyse the product. Keep in mind that Phusion products are blunt-ended.
Cycling table for Phusion and Phusion Hot Start II
| Stage | Temperature | Time |
|---|---|---|
| Initial denaturation | 98 °C | 30 s (up to 3 min for some templates) |
| Denaturation (each cycle) | 98 °C | 5-10 s |
| Annealing (3-step cycling) | Primer-dependent | 10-30 s |
| Extension (each cycle) | 72 °C | 15-30 s per kb |
| Cycles | 25-35 | |
| Final extension | 72 °C | 5-10 min |
| Hold | 4 °C | Indefinite |
The cycling can be 3-step (annealing at a separate temperature) or 2-step, where annealing and extension are combined at 72 °C. The vendor recommends 2-step cycling for primer pairs with a high melting temperature. The vendor also states the extension time must not exceed 1 minute per kb.
Worked example: extension time from amplicon length
The vendor’s rule is 15 s per kb for low-complexity templates (plasmid, lambda, BAC) and 30 s per kb for high-complexity genomic DNA. Applying it by simple arithmetic: a 3 kb product from a plasmid needs about 45 s of extension per cycle (3 × 15), while the same 3 kb product from genomic DNA needs about 90 s (3 × 30). Both stay inside the 1 minute per kb ceiling.
Phusion habits that differ from Taq protocols
Most searchers arrive with a generic protocol for PCR written around Taq. These are the points where the vendor’s Phusion instructions differ in kind, not just in number.
| Topic | What the vendor states for Phusion |
|---|---|
| Denaturation | 98 °C, for 5-10 s per cycle. If yield is low, the vendor lists a denaturation temperature that is too low as a cause. |
| Annealing temperature | May differ significantly from that of Taq-based polymerases. Use the Thermo Scientific Tm calculator. A gradient can extend up to the extension temperature. |
| Combined annealing and extension | 2-step cycling, with no separate annealing step, is recommended for high-Tm primer pairs. |
| Extension time | 15-30 s per kb, never over 1 min per kb. |
| Product ends | Blunt, because of 3′-5′ exonuclease activity. For TA cloning, purify away all Phusion before adding A overhangs with Taq, or the overhangs are degraded. See TA cloning and TOPO cloning. |
| DMSO | Lowers the effective annealing temperature: the vendor cites a report that 10% DMSO lowers it by 5.5-6.0 °C. |
Which Phusion product matches which protocol
The cycling table above applies to Phusion and Phusion Hot Start II. Two other family members use different programs. The table below is as published by the vendor in its lab aid (COL32688 0918); the fidelity figures are the vendor’s own comparison against Taq.
| Product (catalogue no.) | Hot start | Fidelity vs Taq (vendor-stated) | Notes from the vendor |
|---|---|---|---|
| Phusion (F530S) | No | 52x | Target up to 16 kb genomic or 20 kb phage DNA; not tolerant of dUTP |
| Phusion Hot Start II (F549S) | Yes | 52x | Uses the cycling table above |
| Phusion Flash (F548S) | Yes | 25x | 15 s per kb extension; different program, see below |
| Phusion U Hot Start (F555S) | Yes | 25x | dUTP tolerant; target up to 7.5 kb genomic |
Phusion Flash program, per the vendor: initial denaturation 98 °C for 10 s; denaturation 98 °C for 0-1 s; 30 cycles; annealing 50-72 °C for 5 s; extension 15 s per kb; final extension 72 °C for 1 min. If your template or dNTP mix contains uracil, the vendor directs you to Phusion U Hot Start.
What can go wrong
Symptom-to-fix map
| Symptom | Levers the vendor lists |
|---|---|
| No product or low yield | Check pipetting; titrate template; increase extension time or cycle number; optimise annealing; confirm denaturation is at 98 °C or higher; titrate DMSO from 2% to 8%; check the primers. |
| Smeared product | Shorten the extension; use fewer cycles; raise the annealing temperature or use 2-step cycling; lower the primer concentration. |
| Low-molecular-weight bands | Raise the annealing temperature; lower the primer concentration; shorten the extension; titrate the template; redesign the primers. |
HF buffer or GC buffer, and how much DMSO?
The vendor reports an error rate of 4.4 × 10-7 in HF buffer and 9.5 × 10-7 in GC buffer, measured in a lacI-based assay. In HF buffer that is stated to be about 50-fold lower than Taq and 6-fold lower than Pfu. For GC-rich templates the vendor suggests 3% DMSO and varying it in 2% steps. It does not recommend DMSO for templates with very low GC content or amplicons longer than 20 kb. Because DMSO lowers the effective annealing temperature, re-optimise annealing whenever you change it.
Record what you ran
Write down the product and catalogue number, lot, buffer (HF or GC), template type and amount, primer concentration, DMSO percentage, annealing temperature, extension time per kb and cycle number. These are the variables the vendor’s troubleshooting steps adjust, so a record of them lets you change one at a time. For mutagenesis workflows that use a high-fidelity polymerase, see site-directed mutagenesis.
When to get professional help
If a titrated protocol still gives no product, contact the vendor’s technical support with your record sheet, since they can compare it against the product’s specifications. Switch products when the template does not fit the one you chose: for example, Phusion U Hot Start when uracil is present, or a different family member when the amplicon length or hot-start requirement differs from the table above. These products are for research use and are not for diagnostic procedures.
FAQ
What is the Phusion PCR protocol?
It is the reaction setup and cycling program the vendor specifies for Phusion DNA polymerases: 0.5 µM primers, 200 µM dNTPs, 98 °C denaturation, 25-35 cycles, and 15-30 s per kb extension at 72 °C.
What extension time should a PCR protocol with Phusion use?
Use 15 s per kb for low-complexity DNA such as plasmid, lambda or BAC, and 30 s per kb for high-complexity genomic DNA. The vendor states not to exceed 1 minute per kb.
What annealing temperature should I use?
The vendor states that the optimal annealing temperature for Phusion may differ significantly from that for Taq-based polymerases, and recommends its Tm calculator. For high-Tm primer pairs it recommends 2-step cycling with combined annealing and extension at 72 °C.
Should I use HF buffer or GC buffer?
HF buffer is the default. The vendor recommends GC buffer for GC-rich, difficult or long templates, with a higher stated error rate than HF buffer (9.5 × 10-7 against 4.4 × 10-7).
Can I clone a Phusion product by TA cloning?
Not directly: Phusion products are blunt-ended. The vendor states that all Phusion must be purified away before adding A overhangs with Taq, otherwise the overhangs are degraded.
Sources
- Thermo Fisher Scientific. Phusion High-Fidelity PCR Master Mix, product information, Pub. MAN0012771 Rev. C.00 (27 June 2018). https://documents.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012771_Phusion_HiFi_PCR_MasterMix_100rxn_UG.pdf. Date checked: 2026-10-29.
- Thermo Fisher Scientific. Phusion High-Fidelity DNA Polymerases lab aid, COL32688 0918 (2018). https://documents.thermofisher.com/TFS-Assets/BID/Reference-Materials/phusion-dna-polymerases-labaid.pdf. Date checked: 2026-10-29.
All concentrations, times and fidelity figures above are as stated by the vendor for its products.
