"
Team:Tuebingen/Notebook/Protocols/3a-assembly
From 2013.igem.org
(Difference between revisions)
| Line 19: | Line 19: | ||
<p> </p> | <p> </p> | ||
| - | |||
| - | |||
<h3>Reagents</h3> | <h3>Reagents</h3> | ||
| Line 30: | Line 28: | ||
<tr> | <tr> | ||
| - | <td style="text-align: center"> | + | <td style="text-align: center">2.5 µL</td> |
| - | <td> | + | <td><a href="https://www.neb.com/products/b7002-nebuffer-2">10x NEBuffer 2</a></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td style="text-align: center"> | + | <td style="text-align: center">0.25 µL</td> |
| - | <td> | + | <td>100x BSA</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td style="text-align: center"> | + | <td style="text-align: center">250 - 300 ng</td> |
| - | <td> | + | <td>Plasmid DNA</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td style="text-align: center"> | + | <td style="text-align: center">0.5 µL</td> |
| - | <td> | + | <td>EcoRI or XbaI</td> |
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td style="text-align: center">0.5 µL</td> | ||
| + | <td>PstI or SpeI</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td style="text-align: center">to 25 µL</td> | ||
| + | <td>Aqua dest.</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td style="text-align: center">1/10 vol</td> | ||
| + | <td>10X Antarctic Phosphatase</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
| Line 53: | Line 66: | ||
<h3>Procedure</h3> | <h3>Procedure</h3> | ||
| - | <p> | + | <p>Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night. On the next day, heat inactivate restriction enzymes at 80°C for 20 min. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions. Heat inactivate phosphatase at 70 °C for 5 min.</p> |
| + | |||
| + | <p>Run <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/gelelectrophoresis">gel</a> and continue with <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/gelextraction">gelextraction</a>.</p> | ||
</div> | </div> | ||
| + | |||
<!-- FOOTER --- IMPORTANT FOR NAVMENU-LENGTH!!! --> | <!-- FOOTER --- IMPORTANT FOR NAVMENU-LENGTH!!! --> | ||
<div id="TueFooter" style="width: 57em; height: 0.5em; background-color: transparent; position: relative; float: right;"></div> | <div id="TueFooter" style="width: 57em; height: 0.5em; background-color: transparent; position: relative; float: right;"></div> | ||
Revision as of 01:35, 4 October 2013
3A-Assembly
Back to Protocols
Reagents
| 2.5 µL | 10x NEBuffer 2 |
| 0.25 µL | 100x BSA |
| 250 - 300 ng | Plasmid DNA |
| 0.5 µL | EcoRI or XbaI |
| 0.5 µL | PstI or SpeI |
| to 25 µL | Aqua dest. |
| 1/10 vol | 10X Antarctic Phosphatase |
Procedure
Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night. On the next day, heat inactivate restriction enzymes at 80°C for 20 min. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions. Heat inactivate phosphatase at 70 °C for 5 min.
Run gel and continue with gelextraction.
