Team:UNITN-Trento/Protocols
From 2013.igem.org
(Difference between revisions)
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</table> | </table> | ||
</html>|Phusion-PCR}} | </html>|Phusion-PCR}} | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|OneTaq + Phu PCR|<html> | ||
+ | 1) Add the following components in a sterile microtube on ice: | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan="2"> | ||
+ | Reaction Mix | ||
+ | </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>5x One Taq Buffer</th> | ||
+ | <td>10 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Fwd Primer</th> | ||
+ | <td>1 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Rev Primer</th> | ||
+ | <td>1 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>10 mM dNTP's</th> | ||
+ | <td>1 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>One Taq</th> | ||
+ | <td>0.25 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Phusion</th> | ||
+ | <td>0.3 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Template DNA</th> | ||
+ | <td>50-100 ng</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>H20</th> | ||
+ | <td>up to 50 µl</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | 2) Suggested reaction parameters: | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Cycle Step</th> | ||
+ | <th>Cycles</th> | ||
+ | <th>Temp [°C]</th> | ||
+ | <th>Time [s]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Inital denaturation</td> | ||
+ | <td>1</td> | ||
+ | <td>94</td> | ||
+ | <td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Denaturation</td> | ||
+ | <td>30</td> | ||
+ | <td>94</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Annealing</td> | ||
+ | <td></td> | ||
+ | <td>60 *</td> | ||
+ | <td>60</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Extension</td> | ||
+ | <td></td> | ||
+ | <td>68</td> | ||
+ | <td>60 s/Kbase</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Final Extension</td> | ||
+ | <td>1</td> | ||
+ | <td>68</td> | ||
+ | <td>300</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Hold</td> | ||
+ | <td>1</td> | ||
+ | <td>4</td> | ||
+ | <td>∞</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html>|OneTaq-Phu-PCR}} | ||
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Illustra GFX PCR DNA and Gel Band Purification Kit|<html> | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Illustra GFX PCR DNA and Gel Band Purification Kit|<html> |
Revision as of 09:23, 13 August 2013