Team:Groningen/protocols/Ligation
From 2013.igem.org
(Difference between revisions)
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</ul> | </ul> | ||
+ | <h3>Reaction:</h3> | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <th>Component</th> | ||
+ | <th>20µl</th> | ||
+ | <th>Final concentration</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>MilliQ water</td> | ||
+ | <td>up to 20µl</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>10 ligation buffer</td> | ||
+ | <td>2µl</td> | ||
+ | <td>1x</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Vector</td> | ||
+ | <td>xµl</td> | ||
+ | <td>20-100ng</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Insert</td> | ||
+ | <td>xµl</td> | ||
+ | <td>3:1 molar ratio over vector</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>T4 DNA ligase 5U/µl</td> | ||
+ | <td>2µl</td> | ||
+ | <td>0.5U/µl</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
<h3>Reaction:</h3> | <h3>Reaction:</h3> | ||
<table border = "1"> | <table border = "1"> |
Revision as of 15:06, 27 July 2013
Ligation
Materials:
- MQ water
- 1.5ml tubes
- Ligation buffer
- Ligase
- Insert
- Vector
Reaction:
Component | 20µl | Final concentration |
---|---|---|
MilliQ water | up to 20µl | |
10 ligation buffer | 2µl | 1x |
Vector | xµl | 20-100ng |
Insert | xµl | 3:1 molar ratio over vector |
T4 DNA ligase 5U/µl | 2µl | 0.5U/µl |
Reaction:
Component | 20μl reaction | Final concentration |
H2O | up to 20μl | |
10x Ligation buffer | 2μl | 1x |
Vector | 1μl | 20-100μg |
Insert | 1μl | 1:1 molar ratio |
T4 DNA Ligase 5U/μl | 0.2μl | 1U/μl |
All the reagents are added following the order listed in the table above.
After the reaction is ready mix the content of the tube and spin it down.
The tubes are incubated for at room temperature (~22°C) for 1h.
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf