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Team:Macquarie Australia/Protocols/ligations
From 2012.igem.org
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{{:Team:Macquarie_Australia/Template/MQ12}} | {{:Team:Macquarie_Australia/Template/MQ12}} | ||
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<center><h1>Ligation Procedure</h1></center> | <center><h1>Ligation Procedure</h1></center> | ||
<p>A reaction mixture was prepared containing,<br> | <p>A reaction mixture was prepared containing,<br> | ||
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<td>2 µL</td></tr> | <td>2 µL</td></tr> | ||
<tr><td>Destination Plasmid</td> | <tr><td>Destination Plasmid</td> | ||
| - | <td> | + | <td>1 µL</td></tr> |
<tr><td>10X T4 DNA ligase buffer</td> | <tr><td>10X T4 DNA ligase buffer</td> | ||
<td>2 µL</tr></td> | <td>2 µL</tr></td> | ||
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<td>1 µL</td></tr> | <td>1 µL</td></tr> | ||
<tr><td>H<sub>2</sub>O</td> | <tr><td>H<sub>2</sub>O</td> | ||
| - | <td> | + | <td>12 µL</td></tr> |
</table></center> | </table></center> | ||
| - | + | </p> | |
| - | + | <p>We incubated each ligation mix at 30°C for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.</p> | |
| + | <p>4 µL of the ligation product was transformed into 100 µL of competent <i>E. coli</i>, the transformants were then incubated for one hour.</p> | ||
</html> | </html> | ||
Latest revision as of 13:08, 25 September 2012
Ligation Procedure
A reaction mixture was prepared containing,
| Element | Volume |
| Upstream Digestion part | 2 µL |
| Downstream Digestion part | 2 µL |
| Destination Plasmid | 1 µL |
| 10X T4 DNA ligase buffer | 2 µL |
| T4 DNA ligase | 1 µL |
| H2O | 12 µL |
We incubated each ligation mix at 30°C for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.
4 µL of the ligation product was transformed into 100 µL of competent E. coli, the transformants were then incubated for one hour.
