Thermo Scientific Phusion High-F idelity DNA Polymerase offers extreme performance for all major PCR applications. Incorporating an exciting technology, Phusion ® DNA Polymerase brings together a novel Pyrococcus -like enzyme with a processivity-enhancing domain. The Phusion DNA Polymerase generates long templates with an accuracy and speed previously unattainable with a single enzyme, even on the most difficult templates. The extreme fidelity makes Phusion DNA Polymerase a superior choice for cloning. Using a lac I-based method modified from previous studies 1, the error rate of Phusion DNA Polymerase in Phusion HF Buffer is determined to be 4.4 x 10-7, which is approximately 50-fold lower than that of Thermus aquaticus DNA polymerase , and 6-fold lower than that of Pyrococcus furiosus DNA polymerase .
Phusion 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 polymerase is also capable of amplifying long amplicons such as the 7.5 kb genomic and 20 kb l DNA used in Thermo Fisher Scientific quality control assays.
F-530S/L, 100 U/500 U
2. Package information
* Both 5x Phusion HF Buffer and 5x Phusion GC Buffer provide 1.5 mM MgCl 2 in
final reaction concentration.
Material safety data sheet (MSDS) is available at www.thermoscientific/fzmsds.
3. Guidelines for using Phusion DNA Polymerase
Carefully mix and centrifuge all tubes before opening to ensure homogeneity and improve recovery. PCR reactions should be set up on ice. Prepare a master mix for the appropriate number of samples to be amplified. Phusion DNA Polymerase should be pipetted carefully and gently as the high glycerol content (50 %) in the storage buffer may otherwise lead to pipetting errors. It is critical that the Phusion 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.
Due to the nature of Phusion DNA Polymerase, the optimal reaction conditions may differ from PCR protocols for standard DNA polymerases. Due to the high salt concentration in the reaction buffer, Phusion DNA Polymerase tends to work better at elevated denaturation and annealing temperatures. Please pay special attention to the conditions listed in section 5 when running your reactions. Following the guidelines will ensure optimal enzyme performance.
Thermo Scientific
Phusion High-Fidelity DNA Polymerase
Product Information
Table 1. Pipetting instructions: add items in this order.
* Optionally 5x Phusion GC Buffer can be used. See section 4.2. for details
** 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.
*** 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.
Table 2. Cycling instructions.4. Notes about reaction components
4.1 enzyme
The optimal amount of enzyme depends on the amount of template and the length of the PCR product. Usually 1 unit of Phusion 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 difficulty. It is not recommended to exceed 2 U/50 µl (0.04 U/µl), especially for amplicons that are > 5kb.When cloning fragments amplified with Phusion 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 or DreamTaq DNA Polymerase, for example. However, before ad
ding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully. Any remaining Phusion DNA Polymerase will degrade the A overhangs, creating blunt ends again. A detailed protocol for TA cloning of DNA fragments amplified with any of the Phusion DNA polymerases can be found on our website (www.thermoscientific/pcrcloning).
4.2 Buffers
Two buffers are provided with the enzyme: 5x Phusion HF Buffer and 5x Phusion GC Buffer. The error rate of Phusion 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 DNA Polymerase on some difficult 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 need to be free of detergents, detergent-free reaction buffers are available for Phusion DNA Polymerases. 4.3 mg 2+ and dNTP
The concentration of Mg 2+ is critical since Phusion 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 buffer 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 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 DNA Polymerase there is no advantage of increasing dNTP concentrations. For optimal results always use 200 µM of each dNTP .
4.4 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.
4.5 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 DNA Polymerase.
If high DMSO concentration is used, the annealing temperature must be decreased, as DMSO affects the melting point of the primers. It has been reported that 10 % DMSO decreases the annealing temperature by 5.5–6.0°C 2.
5. Notes about cycling conditions
5.1 Initial denaturation
Denaturation should be performed at 98°C. Due to the high
thermostability of Phusion 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.
5.2 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.
5.3 Primer annealing
The optimal annealing temperature for Phusion DNA Polymerase
may differ significantly from that of Taq-based polymerases.
Always use the Tm calculator and instructions on our website
(www.thermoscientific/pcrwebtools) to determine the Tm
values of primers and optimal annealing temperature.
真如中学The Phusion DNA Polymerase has the ability to stabilize primer-
template hybridization. As a basic rule, for primers > 20 nt, anneal
for 10–30 seconds at a Tm +3°C of the lower Tm primer. For primers
≤ 20 nt, 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). 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.
5.4 extension
The extension should be performed at 72°C. Extension time depends
on amplicon length and complexity. F or 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.
6. Troubleshooting
7. Component specifications
7.1 Phusion High-Fidelity DNA Polymerase (F-530)
Thermostable Phusion DNA Polymerase is purified from li
strain expressing the cloned Phusion DNA Polymerase gene. Phusion
DNA Polymerase possesses the following activities: 5´→3´ DNA
polymerase activity and 3´→5´ exonuclease activity. Phusion DNA
Polymerase is free of contaminating endo- and exonucleases. Storage buffer: 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 acid-insoluble form at 74°C in 30 minutes under the stated assay conditions.
Unit assay conditions: Incubation buffer: 25 mM TAPS-HCl, pH 9.3 (at 25°C), 50 mM KCl, 2 mM MgCl
2
, 1 mM b-mercaptoethanol, 100 µM dCTP, 200 µM each dATP, dGTP, dTTP.
罗尔
中值定理Incubation procedure: 20 µg activated calf thymus DNA and 0.5 µCi [a-32P] dCTP are incubated with 0.1 units of DNA polymerase in 50 µl incubation buffer at 74°C for 10 minutes. The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation.
DNA amplification assay: Performance in PCR is tested by the amplification of 7.5 kb fragment of genomic DNA and 20 kb fragment of l DNA.
exonuclease contamination assay: Incubation of 10 U for 4 hours at 72°C in 50 µl assay buffer with 1 µg sonicated [3H]-ssDNA (2x105 cpm/µg) released < 1% of radioactivity.
endonuclease contamination assay: No endonuclease activity was observed after incubation of 10 U of DNA polymerase with 1 µg of l DNA in assay buffer at 72°C for 4 hours.
7.2 5x Phusion HF Buffer (F-518)
The 5x Phusion HF Buffer contains 7.5 mM MgCl
2
, which provides 1.5 mM MgCl
2
in final reaction conditions.
7.3 5x Phusion GC Buffer (F-519)
The 5x Phusion GC Buffer contains 7.5 mM MgCl
2
, which provides 1.5 mM MgCl
2
in final reaction conditions.
郭杰自杀7.4 50 mm mgCl
2
Solution (F-510mG)
Both Phusion Buffers 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.
7.5 Dimethyl sulfoxide DmSo, 100 % (F-515)
edk2Note: The freezing point of DMSO is 18–19°C, so it does not melt on ice.
8. references
1. Frey M. & Suppmann B. (1995) Biochemica 2: 34–35.
2. Chester N. & Marshak D.R. (1993) Analytical Biochemistry
209: 284–290.Shipping and storage
Phusion DNA Polymerase is shipped on gel ice. Upon arrival, store the components at -20°C.
Technical support:
US: ics@thermofisher
Europe, Asia, Rest of World:
Web: www.thermoscientific/phusion
Tm-calculator: www.thermoscientific/pcrwebtools
Product use limitation
This product has been developed and is sold exclusively for research purposes and in vitro use only. This product has not been tested for use in diagnostics or drug development, nor are they suitable for administration to humans or animals.
Notice to Purchaser: Limited License
德鲁兹
The purchase price of this product includes a limited, non-transferable license under national patents from EP 0 547 920 B1, owned by New England Biolabs, Inc., to use this product. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product.
The purchase price of this product includes a limited, nontransferable license under U.S. and foreign patents owned by BIO-RAD Laboratories, Inc., to use this product. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product.
Designed and manufactured according to certified ISO9001:2008 processes.
© 2011 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
v1_07.2011
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