Phusion
™
High–Fidelity DNA Polymerase
Catalog Numbers F530S and F530L
Pub. No. MAN0012393 Rev. E.0
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Product description
The Thermo Scientific
™
Phusion
™
High–Fidelity DNA Polymerase oers high performance for all major PCR applications. The Phusion
™
High–Fidelity DNA Polymerase brings together a Pyrococcus–like enzyme with a processivity–enhancing domain. The Phusion
™
High–
Fidelity DNA Polymerase generates long amplicons with accuracy and speed, even on the most dicult templates. The high fidelity makes
the Phusion
™
High–Fidelity DNA Polymerase a superior choice for cloning. Using a lacI–based method modified from previous studies (see
reference 1 on page 4), the error rate of Phusion
™
High–Fidelity DNA Polymerase in Phusion
™
HF Buer is determined to be 4.4 × 10
-7
,
which is approximately 50 fold lower than that of the Thermus aquaticus DNA polymerase, and 6‑fold lower than that of the Pyrococcus
furiosus DNA polymerase.
The Phusion
™
High–Fidelity DNA Polymerase possesses the following activities: 5´→3´ DNA polymerase activity and 3´→5´ exonuclease
activity. It generates blunt ends in the amplification products. The Phusion
™
High–Fidelity DNA Polymerase is also capable of amplifying
long amplicons such as the 7.5 kb genomic and 20 kb λ DNA.
Contents and storage
Component
F530S F530L
Phusion
™
DNA Polymerase, 2 U/µL 100 U
50 µL
500 U
250 µL
5X Phusion
™
HF Buer
[1]
2 × 1.5 mL 6 × 1.5 mL
5X Phusion
™
GC Buer 1.5 mL 2 × 1.5 mL
50 mM MgCl
2
solution 1.5 mL 2 × 1.5 mL
DMSO 500 µL 500 µL
[1]
Both 5X Phusion
™
HF Buffer and 5X Phusion
™
GC Buffer provide 1.5 mM MgCl
2
in final reaction concentration.
Important notes
• Use 98°C for denaturation (see “Initial denaturation” on page 3 and “Denaturation” on page 3).
• The annealing rules are dierent from many common DNA polymerases (such as Taq DNA polymerases). Read “Primer annealing” on
page 3 carefully.
• Use 15–30 s/kb for extension. Do not exceed 1 min/kb (see “Extension” on page 4).
• Use Phusion
™
High–Fidelity DNA Polymerase at 0.5–1.0 U per 50 µL reaction volume. Do not exceed 2 U/50 µL (see “Enzyme” on
page 3).
• Use 200 µM of each dNTP. Do not use dUTP (see “Mg2+ and dNTP” on page 3).
• Phusion
™
High–Fidelity DNA Polymerase produce blunt end DNA products.
Recommended protocol
• PCR reactions should be set up on ice.
• Prepare a master mix for the appropriate number of samples to be amplified.
PRODUCT INFORMATION SHEET
For Research Use Only. Not for use in diagnostic procedures.
• The Phusion
™
High–Fidelity DNA Polymerase should be pipetted carefully and gently as the high glycerol content (50%) in the
storage buer may otherwise lead to pipetting errors.
• Due to the nature of the Phusion
™
High–Fidelity DNA Polymerase, the optimal reaction conditions may dier from PCR protocols for
standard DNA polymerases.
• Due to the high salt concentration in the reaction buer, the Phusion
™
High–Fidelity DNA Polymerase tends to work better at elevated
denaturation and annealing temperatures.
• Follow the conditions listed in “Notes about cycling conditions” on page 3 when running your reactions.
1. Prepare PCR reactions. Add the following components in the order listed in the following table.
Note:
·
It is critical that the Phusion
™
High–Fidelity DNA Polymerase is the last component added to the PCR mixture, since the enzyme
exhibits 3´→5´ exonuclease activity that can degrade primers in the absence of dNTPs.
·
Carefully mix and centrifuge all tubes before opening to ensure homogeneity and improve recovery.
Component
20 µL rxn 50 µL rxn Final conc.
H
2
O add to 20 µL add to 50 µL
5X Phusion
™
HF Buer
[1]
4 µL 10 µL 1X
10 mM dNTPs 0.4 µL 1 µL 200 µM each
Forward primer
[2]
X µL X µL 0.5 µM
Reverse primer
[2]
X µL X µL 0.5 µM
Template DNA X µL X µL
(DMSO
[3]
, optional) (0.6 µL) (1.5 µL) (3%)
Phusion
™
High–Fidelity DNA Polymerase 0.2 µL 0.5 µL 0.02 U/µL
[1]
Optionally 5X GC Buffer can be used. See “Buffers” on page 3 for details.
[2]
The recommendation for final primer concentration is 0.5 µM, but it can be varied in a range of 0.2–1.0 µM, if needed.
[3]
Addition of DMSO is recommended for GC-rich amplicons. DMSO is not recommended for amplicons with very low GC % or amplicons that are > 20 kb.
2. Run the following program.
Cycle step
2–step protocol 3–step protocol
Cycles
Temp. Time Temp. Time
Initial Denaturation 98°C 30 s 98°C 30 s 1
Denaturation 98°C 5–10 s 98°C 5–10 s 25–35
Annealing
[1,2]
– – X°C 10–30 s
Extension
[3,4]
72°C 15–30 s/kb 72°C 15–30 s/kb
Final extension 72°C 5–10 min 72°C 5–10 min 1
Hold 4°C Hold 4°C Hold Hold
[1]
See “Primer annealing” on page 3.
[2]
For the 2–step protocol, there is no annealing step.
[3]
See “Extension” on page 4.
[4]
For the 2–step protocol, annealing and extansion are performed at the same temperature.
2 Thermo Scientific
™
Phusion
™
High–Fidelity DNA Polymerase Product Information Sheet
Notes about reaction components
Enzyme
The optimal amount of enzyme depends on the amount of template and the length of the PCR product. Usually 1 unit of Phusion
™
High–Fidelity DNA Polymerase per 50 µL reaction volume gives good results, but the optimal amount can range from 0.5 to 2 units
per 50 µL reaction depending on amplicon length and diculty. It is not recommended to exceed 2 U/50 µL (0.04 U/µL), especially for
amplicons that are > 5kb.
Buers
Two buers are provided with the enzyme: 5X Phusion
™
HF Buer and 5X Phusion
™
GC Buer. The error rate of Phusion
™
High–Fidelity
DNA Polymerase in HF Buer (4.4 × 10
-7
) is lower than that in GC Buer (9.5 × 10
-7
). Therefore, the HF Buer should be used as the
default buer for high-fidelity amplification. However, GC Buer can improve the performance of Phusion
™
High–Fidelity DNA Polymerase
on some dicult or long templates, such as GC–rich templates or those with complex secondary structures. For applications such as
microarray or DHPLC, where the DNA templates must be free of detergents, detergent–free reaction buers are available for Phusion
™
High–Fidelity DNA Polymerases (#F–520L, #F–521L).
Mg
2+
and dNTP
The concentration of Mg
2+
is critical since Phusion
™
High–Fidelity DNA Polymerase is a magnesium dependent enzyme. Excessive Mg
2+
stabilizes the DNA double strand and prevents complete denaturation of DNA. Excess Mg
2+
can also stabilize spurious annealing of
primers to incorrect template sites and decrease specificity. Conversely, inadequate Mg
2+
may lead to lower product yield. The optimal
Mg
2+
concentration also depends on the dNTP concentration, the specific template DNA and the sample buer composition. In general,
the optimal Mg
2+
concentration is 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 Mg
2+
optimum may be shifted to higher concentrations. If further optimization is needed,
increase Mg
2+
concentration in 0.2 mM steps.
High quality dNTPs should be used for optimal performance with Phusion
™
High–Fidelity DNA Polymerase. The polymerase cannot read
dUTP-derivatives or dITP in the template strand so the use of these analogues or primers containing them is not recommended. Due to
the high processivity of Phusion
™
High–Fidelity DNA Polymerase there is no advantage of increasing dNTP concentrations. For optimal
results always use 200 µM of each dNTP.
Template
General guidelines for low complexity DNA (e.g. plasmid, lambda or BAC DNA) are: 1 pg–10 ng per 50 µL reaction volume. For high
complexity genomic DNA, the amount of DNA template should be 50–250 ng per 50 µL reaction volume. If cDNA synthesis reaction
mixture is used as a source of template, the volume of the template should not exceed 10% of the final PCR reaction volume.
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 the amount of DMSO should be increased in 2% increments. In some cases
DMSO may also be required for supercoiled plasmids to relax for denaturation. Other PCR additives such as formamide, glycerol, and
betaine are also compatible with Phusion
™
High–Fidelity DNA Polymerase.
If high DMSO concentration is used, the annealing temperature must be decreased, as DMSO aects the melting point of the primers. It
has been reported that 10% DMSO decreases the annealing temperature by 5.5–6.0°C (see reference 2 on page 4).
Notes about cycling conditions
Initial denaturation
Denaturation should be performed at 98°C. Due to the high thermostability of Phusion
™
High–Fidelity DNA Polymerase even higher
than 98°C denaturation temperatures can be used. We recommend a 30 second initial denaturation at 98°C for most templates. Some
templates may require longer initial denaturation time and the length of the initial denaturation time can be extended up to 3 minutes.
Denaturation
Keep the denaturation time 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
The optimal annealing temperature for Phusion
™
High–Fidelity DNA Polymerase may dier significantly from that of Taq–based
polymerases. Always use the Tm calculator and instructions on our website (www.thermofisher.com/tmcalculator) to determine the
Tm values of primers and optimal annealing temperature.
A 2-step protocol is recommended when primer Tm values are at least 69°C (> 20 nt) or 72°C (≤ 20 nt) when calculated with our Tm
calculator. In the 2-step protocol the combined annealing/extension step should be performed at 72°C even when the primer Tm is >
72°C.
Thermo Scientific
™
Phusion
™
High–Fidelity DNA Polymerase Product Information Sheet 3
Extension
The extension should be performed at 72°C. Extension time depends on amplicon length and complexity. For low complexity DNA (e.g.
plasmid, lambda or BAC DNA) use an extension time of 15 seconds per 1 kb. For high complexity genomic DNA 30 seconds per 1 kb is
recommended. For some cDNA templates, the extension time can be increased up to 40 seconds per 1 kb to obtain optimal results.
Recommendations for cloning
When cloning fragments amplified with Phusion
™
High–Fidelity 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 Thermo Scientific
™
Taq DNA Polymerase (#EP0402),
for example. Incubate purified PCR product with 1x Taq buer, 2.5 mM MgCl
2
, 0.2 mM dATP and 1 U Taq DNA polymerase in 10 µL
reaction mixture up to 30 min at 72°C. Before adding the overhangs it is very important to remove all the Phusion
™
High–Fidelity DNA
Polymerase by carefully purifying the PCR product, for example using Thermo Scientific
™
GeneJET
™
PCR Purification Kit (#K0701). Any
remaining Phusion
™
High–Fidelity DNA Polymerase will degrade the A overhangs, creating blunt ends again.
Component specifications
Phusion
™
High–Fidelity DNA Polymerase (F-530)
Thermostable Phusion
™
High–Fidelity DNA Polymerase is purified from an E.coli strain expressing the cloned Phusion
™
High–Fidelity DNA
Polymerase gene. Phusion
™
High–Fidelity DNA Polymerase possesses the following activities: 5´→3´ DNA polymerase activity and 3´→5´
exonuclease activity.
Storage buer: 20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, stabilizers, 200 µg/mL BSA and 50% glycerol.
Unit definition: One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTPs into a polynucleotide fraction at
74°C in 30 min.
Enzyme activity is assayed in the following mixture:
25 mM TAPS-HCl, pH 9.3 (at 25°C), 50 mM KCl, 2 mM MgCl
2
, 1 mM β-mercaptoethanol, 0.75 mM activated salmon milt DNA, 100 µM
dTTP, 200 µM each dATP, dGTP, dCTP, 0.4 MBq/ml [
3
H] dTTP.
5X Phusion
™
HF buer (F-518)
The 5X Phusion
™
HF Buer contains 7.5 mM MgCl
2
, which provides 1.5 mM MgCl
2
in final reaction conditions.
5X GC buer (F-519)
The 5X GC Buer contains 7.5 mM MgCl
2
, which provides 1.5 mM MgCl
2
in final reaction conditions.
50 mM MgCl
2
solution (F-510MG)
Both Buers supply 1.5 mM MgCl
2
at final reaction conditions. If higher MgCl
2
concentrations are desired, use 50 mM MgCl
2
solution to
increase the MgCl
2
titer. Using the following equation, you can calculate the volume of 50 mM MgCl
2
needed to attain the final MgCl
2
concentration: [desired mM Mg]–[1.5 mM] = µL to add to a 50 µL reaction.
For example, to increase the MgCl
2
concentration to 2.0 mM, add 0.5 µL of the 50 mM MgCl
2
solution. Because the PCR reactions can be
quite sensitive to changes in the MgCl
2
concentration, it is recommended that the 50 mM MgCl
2
stock solution is diluted 1:5 (to 10 mM) to
minimize pipetting errors.
Dimethyl sulfoxide DMSO, 100% (F-515)
Note: The freezing point of DMSO is 18–19°C, so it does not melt on ice.
References
1. Frey M. & Suppmann B. (1995) Biochemica 2: 34–35.
2. Chester N. & Marshak D.R. (1993) Analytical Biochemistry 209: 284–290.
Troubleshooting and FAQs
Visit our online FAQ database for tips and tricks for conducting your experiment, troubleshooting information, and FAQs. The online FAQ
database is frequently updated to ensure accurate and thorough content.
• For troubleshooting information and FAQs for this product: https://www.thermofisher.com/phusionhifidnapolfaqs
• To browse the database and search using keywords: thermofisher.com/faqs
4 Thermo Scientific
™
Phusion
™
High–Fidelity DNA Polymerase Product Information Sheet
Documentation and support
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The information in this guide is subject to change without notice.
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Revision history: Pub. No. MAN0012393
Revision Date Description
E.0 9 July 2021 Corrected skus in contents and storage table from F553S and F553L to F530S and F530L.
D.0 28 January 2020 Moved troubleshooting content to thermofisher.com.
C.0 27 June 2018 Baseline for revision.
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