Fusion DNA Polymerase
Description
Introduction
Biotool™ Fusion DNA Polymerase is the superior enzyme in this category for robust PCR with extreme fidelity, featuring 50x error rate lower than Taq polymerase and 15 seconds/kb extension. Biotool TM Fusion DNA Polymerase possesses 5´→ 3´polymerase activity, 3´ → 5´exonuclease activity and will generate blunt-ended products. Our suggested applications include cloning, sequencing, genomic DNA amplification and high throughput PCR, etc.
Advantages
 Robust: Maximal success with minimal optimization
 Extreme Fidelity: 50x error rate lower than Taq
 High Speed: 10x faster than Pfu
 High Yield: Increased product yield using minimal enzyme
 Versatile: used for routine PCR, or with long, difficult templates
Materials provided
1. Biotool™ Fusion DNA Polymerase 100 U (1U/μl) 
2. 5x Biotool™ Fusion HF buffer (1.5 ml) 
3. 5x Biotool™ Fusion GC buffer (1.5 ml) 
4. 50mM MgCl2 (1.5 ml) 
5. DMSO (100%) (0.5 ml) 
All materials should be stored at -20℃.
Protocol
1. Add the components into sterile thin-walled PCR tubes while mixing gently.
Component
Amount per reaction (μl)
5x Biotool™ Fusion HF or GC buffer
10
dNTP mix (10 mM of each dNTP)
1
DNA template (100ng/μl)
1-2.5
Upstream primer (10 μM)
2.5
Downstream primer (10 μM)
2.5
Biotool™ Fusion Polymerase
1
DMSO (optional)
1.5
Distilled water (dH2O)
to 50
Total reaction volume
50
2. Perform PCR using optimized cycling conditions. Suggested cycling parameters for using Fusion DNA polymerase are provided below. Analyze the PCR amplification products on a 0.7–1.0% (w/v) agarose gel.
Segment
Number of cycles
Temperature ℃
Duration
1
1
98
30 seconds
2
25-30
98
5-10 seconds
Primer Tm – 5
10-30 seconds
72
15-30 seconds / kb
3
1
72
5-10 minutes
D4
1
4
∞
Note:
1. Optimized cycling parameters maynot necessarily be transferable between thermal cyclers. Consult the instrument manufacturer’s recommendations if further optimization of cycling parameters is required.
2. 5x Fusion HF Buffer is recommended as the default buffer for high-fidelity amplification. 5x Fusion GC Buffer is preferred for amplification with difficult templates, such as GC-rich templates or those with secondary structure. Generally, GC buffer should be used in experiments where HF buffer does not work.
3. Mg++ is critical to achieve optimal activity with Fusion DNA Polymerase. The final Mg++ concentration in 1x Fusion HF and GC Buffer is 1.5 mM. Mg++ can be optimized in 0.5 mM increments using the MgCl2 provided.
4. Addition of DMSO (included) may improve the amplification results of difficult targets, such as those with GC-rich sequences or secondary structure. 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 Tm.
5. For primers with annealing temperatures ≥ 72°C, a 2-step protocol is recommended.
Segment
Number of cycles
Temperature ℃
Duration
1
1
98
30 seconds
2
25-30
98
5-10 seconds
72
15-30 seconds / kb
3
1
72
5-10 minutes
4
1
4
∞
Troubleshooting
Please review the following guidance for low yield or unsuccessful PCR: a, high quality or purified DNA templates are preferred to enhance the success of PCR; b, primers’ final concentration can vary from 0.2-1 μM; c, addition of Mg++ or DMSO may enhance the yield of GC-rich sequence amplification; d, Fusion Polymerase concentration may vary from 0.5–2 units /50 μl reaction, but do not exceed 2 units/50 µl reaction, especially for amplification longer than 5 kb; e, GC buffer may enhance the yield for some difficult reactions; f, annealing temperature may need to be optimized; g, extension time can be increased to 30 seconds / kb.
Limited Product Warranty
This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Biotool.com. We shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product.
Limited Label License for Fusion DNA Polymerase Products
Purchase of this product conveys to the purchaser only the non-transferable right to use the product for research use only by the purchaser. No rights are granted to the purchaser hereunder to sell, modify for resale or otherwise transfer this product, either alone or as a component of another product, to any third party. Biotool.com reserves all other rights, and this product may not be used in any manner other than as provided herein. For information on obtaining a license to use this product for purposes other than research, please contact Biotool.com.
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