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<h3>Phusion ® High-Fidelity DNA Polymerase</h3> | <h3>Phusion ® High-Fidelity DNA Polymerase</h3> | ||
− | < | + | <h4>Composition:</h4> |
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<table frame="void"> | <table frame="void"> | ||
<tbody> | <tbody> | ||
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<tr> | <tr> | ||
− | <td> | + | <td>Phusion HF Buffer (5x)</td> |
<td>10 µl</td> | <td>10 µl</td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>10 mM | + | <td>dNTPs (10 mM)</td> |
<td>1 µl</td> | <td>1 µl</td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>10 µM | + | <td>Forward Primer (10 µM)</td> |
<td>2.5 µl</td> | <td>2.5 µl</td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>10 µM | + | <td>Reverse Primer (10 µM)</td> |
<td>2.5 µl</td> | <td>2.5 µl</td> | ||
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<td>DMSO (optional)</td> | <td>DMSO (optional)</td> | ||
− | <td>1.5 µl | + | <td>up to 3% (1.5 µl)</td> |
</tr> | </tr> | ||
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</table> | </table> | ||
+ | <h4>Thermocycling Conditions:</h4> | ||
+ | <table> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | |||
+ | <td align="center"><strong>Temperatur <br /> | ||
+ | </strong></td> | ||
+ | <td align="center"><strong>Time<br /> | ||
+ | </strong></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | |||
+ | <td align="center">98°C </td> | ||
+ | <td>30 seconds </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | |||
+ | <td align="center">98°C<br /> | ||
+ | 45-72°C<br /> | ||
+ | 72°C </td> | ||
+ | <td>5-10 seconds<br /> | ||
+ | 10-30 seconds<br /> | ||
+ | 15-30 seconds per kb </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | |||
+ | <td align="center">72°C </td> | ||
+ | <td>5-10 minutes </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | |||
+ | <td align="center">4-10°C </td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
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</div> | </div> | ||
</div> | </div> | ||
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+ | <div class="sec white"> | ||
+ | <div> | ||
+ | <h3>Kap Hifi Hotstart Ready Mix</h3> | ||
+ | |||
+ | <table frame="void"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td><strong>Component</strong></td> | ||
+ | <td><strong>50 µl Reaction</strong></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Phusion HF Buffer (5x)</td> | ||
+ | |||
+ | <td>10 µl</td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dNTPs (10 mM)</td> | ||
+ | |||
+ | <td>1 µl</td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Forward Primer (10 µM)</td> | ||
+ | |||
+ | <td>2.5 µl</td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Reverse Primer (10 µM)</td> | ||
+ | |||
+ | <td>2.5 µl</td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Template DNA</td> | ||
+ | |||
+ | <td>variable</td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DMSO (optional)</td> | ||
+ | |||
+ | <td>up to 3% (1.5 µl)</td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Nuclease-free water</td> | ||
+ | |||
+ | <td>up to 50 µl</td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Phusion Polymerase</td> | ||
+ | |||
+ | <td>0.5 µl</td> | ||
+ | |||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
</div> | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
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Revision as of 00:32, 9 October 2016
Experimental: Protocols, Methods and Material
Polymerase Chain Reaction
Phusion ® High-Fidelity DNA Polymerase
Composition:
Component | 50 µl Reaction |
Phusion HF Buffer (5x) | 10 µl |
dNTPs (10 mM) | 1 µl |
Forward Primer (10 µM) | 2.5 µl |
Reverse Primer (10 µM) | 2.5 µl |
Template DNA | variable |
DMSO (optional) | up to 3% (1.5 µl) |
Nuclease-free water | up to 50 µl |
Phusion Polymerase | 0.5 µl |
Thermocycling Conditions:
Temperatur |
Time |
98°C | 30 seconds |
98°C 45-72°C 72°C |
5-10 seconds 10-30 seconds 15-30 seconds per kb |
72°C | 5-10 minutes |
4-10°C |
Kap Hifi Hotstart Ready Mix
Component | 50 µl Reaction |
Phusion HF Buffer (5x) | 10 µl |
dNTPs (10 mM) | 1 µl |
Forward Primer (10 µM) | 2.5 µl |
Reverse Primer (10 µM) | 2.5 µl |
Template DNA | variable |
DMSO (optional) | up to 3% (1.5 µl) |
Nuclease-free water | up to 50 µl |
Phusion Polymerase | 0.5 µl |