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Team:Groningen/protocols/Ligation
From 2013.igem.org
(Difference between revisions)
| Line 27: | Line 27: | ||
<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> | ||
| Line 38: | Line 38: | ||
<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 ALIGN=CENTER>2µl</td> | + | <td ALIGN=CENTER>2 µl</td> |
<td>1x</td> | <td>1x</td> | ||
</tr> | </tr> | ||
| Line 56: | Line 56: | ||
<tr> | <tr> | ||
<td>Vector</td> | <td>Vector</td> | ||
| - | <td ALIGN=CENTER>xµl</td> | + | <td ALIGN=CENTER>x µl</td> |
| - | <td>20- | + | <td>20-100 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Insert</td> | <td>Insert</td> | ||
| - | <td ALIGN=CENTER>xµl</td> | + | <td ALIGN=CENTER>x µl</td> |
<td>3:1 molar ratio over vector*</td> | <td>3:1 molar ratio over vector*</td> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td>T4 DNA ligase 5U/µl</td> | <td>T4 DNA ligase 5U/µl</td> | ||
| - | <td ALIGN=CENTER>2µl</td> | + | <td ALIGN=CENTER>2 µl</td> |
<td>0.5U/µl</td> | <td>0.5U/µl</td> | ||
</tr> | </tr> | ||
Revision as of 15:05, 27 September 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 5U/µl | 2 µl | 0.5U/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.
After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min.
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
iGEM 2013 Groningen
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