Team:Groningen/protocols/Ligation
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+ | <h2>Ligation Reaction</h2> | ||
+ | |||
+ | <h5>Materials:</h5> | ||
+ | <ul type="square"> | ||
+ | <li>MQ water</li> | ||
+ | <li>1.5 ml tubes</li> | ||
+ | <li>10x T4 Ligation buffer</li> | ||
+ | <li>T4 Ligase</li> | ||
+ | <li>Insert</li> | ||
+ | <li>Vector</li> | ||
+ | </ul> | ||
+ | |||
+ | <h5>Reaction mixture:</h5> | ||
+ | <table id="normal" width="60%"> | ||
+ | <tr> | ||
+ | <th>Component</th> | ||
+ | <th>20 µl</th> | ||
+ | <th>Final concentration</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>MilliQ water</td> | ||
+ | <td>up to 20 µl</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>10 ligation buffer</td> | ||
+ | <td>2 µl</td> | ||
+ | <td>1x</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Vector</td> | ||
+ | <td>x µl</td> | ||
+ | <td>20-100 ng</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Insert</td> | ||
+ | <td>x µl</td> | ||
+ | <td>3:1 molar ratio over vector*</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>T4 DNA ligase 5 U/µl</td> | ||
+ | <td>2 µl</td> | ||
+ | <td>0.5 U/µl</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
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+ | <p>* When restricted PCR products are ligated together, a 1:1 molar ratio is used. | ||
+ | |||
+ | <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. | ||
+ | |||
+ | |||
+ | <p>[1] http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf | ||
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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