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Team:Groningen/protocols/Ligation
From 2013.igem.org
(Difference between revisions)
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<ul type="square"> | <ul type="square"> | ||
<li>MQ water</li> | <li>MQ water</li> | ||
| - | <li>1. | + | <li>1.5 ml tubes</li> |
<li>10x T4 Ligation buffer</li> | <li>10x T4 Ligation buffer</li> | ||
<li>T4 Ligase</li> | <li>T4 Ligase</li> | ||
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<h5>Reaction mixture:</h5> | <h5>Reaction mixture:</h5> | ||
| - | <table id="normal"> | + | <table id="normal" width="60%"> |
<tr> | <tr> | ||
<th>Component</th> | <th>Component</th> | ||
| - | <th>20µl</th> | + | <th>20 µl</th> |
<th>Final concentration</th> | <th>Final concentration</th> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td>MilliQ water</td> | <td>MilliQ water</td> | ||
| - | <td>up to 20µl</td> | + | <td>up to 20 µl</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 50: | Line 50: | ||
<tr> | <tr> | ||
<td>10 ligation buffer</td> | <td>10 ligation buffer</td> | ||
| - | <td | + | <td>2 µl</td> |
<td>1x</td> | <td>1x</td> | ||
</tr> | </tr> | ||
| Line 56: | Line 56: | ||
<tr> | <tr> | ||
<td>Vector</td> | <td>Vector</td> | ||
| - | <td | + | <td>x µl</td> |
| - | <td>20- | + | <td>20-100 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Insert</td> | <td>Insert</td> | ||
| - | <td | + | <td>x µl</td> |
<td>3:1 molar ratio over vector*</td> | <td>3:1 molar ratio over vector*</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>T4 DNA ligase | + | <td>T4 DNA ligase 5 U/µl</td> |
| - | <td | + | <td>2 µl</td> |
| - | <td>0. | + | <td>0.5 U/µl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| - | < | + | <p>* When restricted PCR products are ligated together, a 1:1 molar ratio is used. |
| - | < | + | |
| - | All the reagents are added following the order listed in the table above. | + | <br>All the reagents are added following the order listed in the table above [1]. |
<br>After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min. | <br>After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min. | ||
| - | <p> | + | |
| - | + | <p>[1] http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf | |
</div> | </div> | ||
Latest revision as of 23:36, 4 October 2013
Ligation Reaction
Materials:
- MQ water
- 1.5 ml tubes
- 10x T4 Ligation buffer
- T4 Ligase
- Insert
- Vector
Reaction mixture:
| Component | 20 µl | Final concentration |
|---|---|---|
| MilliQ water | up to 20 µl | |
| 10 ligation buffer | 2 µl | 1x |
| Vector | x µl | 20-100 ng |
| Insert | x µl | 3:1 molar ratio over vector* |
| T4 DNA ligase 5 U/µl | 2 µl | 0.5 U/µl |
* When restricted PCR products are ligated together, a 1:1 molar ratio is used.
All the reagents are added following the order listed in the table above [1].
After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min.
[1] http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
iGEM 2013 Groningen
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