Line 75: | Line 75: | ||
<td>DMSO (optional)</td> | <td>DMSO (optional)</td> | ||
− | <td>up to 3% | + | <td>up to 3%</td> |
</tr> | </tr> | ||
Line 149: | Line 149: | ||
<tr> | <tr> | ||
− | <td> | + | <td>Master Mix (2x)</td> |
− | <td> | + | <td>25 µl</td> |
</tr> | </tr> | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<tr> | <tr> | ||
<td>Forward Primer (10 µM)</td> | <td>Forward Primer (10 µM)</td> | ||
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</tr> | </tr> | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<tr> | <tr> | ||
<td>Nuclease-free water</td> | <td>Nuclease-free water</td> | ||
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</tr> | </tr> | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
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</div> | </div> | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | |||
Revision as of 00:34, 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% |
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 |
Master Mix (2x) | 25 µl |
Forward Primer (10 µM) | 2.5 µl |
Reverse Primer (10 µM) | 2.5 µl |
Template DNA | variable |
Nuclease-free water | up to 50 µl |