Line 122: | Line 122: | ||
<td>5-10 minutes </td> | <td>5-10 minutes </td> | ||
</tr> | </tr> | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
Line 181: | Line 177: | ||
</tr> | </tr> | ||
+ | </tbody> | ||
+ | </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">95°C </td> | ||
+ | <td>3 minutes </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | |||
+ | <td align="center">98°C<br /> | ||
+ | 55-75°C<br /> | ||
+ | 72°C </td> | ||
+ | <td>20 seconds<br /> | ||
+ | 15 seconds<br /> | ||
+ | 15-60 seconds per kb </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | |||
+ | <td align="center">72°C </td> | ||
+ | <td>1 minute per kb </td> | ||
+ | </tr> | ||
+ | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
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</div> | </div> | ||
</div> | </div> | ||
+ | |||
Revision as of 00:41, 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 |
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 |
Thermocycling Conditions:
Temperatur |
Time |
95°C | 3 minutes |
98°C 55-75°C 72°C |
20 seconds 15 seconds 15-60 seconds per kb |
72°C | 1 minute per kb |