Team:Groningen/protocols/Ligation
From 2013.igem.org
(Difference between revisions)
Line 37: | Line 37: | ||
<table border = "1"> | <table border = "1"> | ||
<tr><td>Component</td> | <tr><td>Component</td> | ||
- | <td> | + | <td>20μl reaction</td> |
<td>Final concentration</td> | <td>Final concentration</td> | ||
</tr> | </tr> | ||
<tr><td>H<sub>2</sub>O</td> | <tr><td>H<sub>2</sub>O</td> | ||
- | <td>up to | + | <td>up to 20μl</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
- | <tr><td> | + | <tr><td>10x Ligation buffer</td> |
- | <td> | + | <td>2μl</td> |
<td>1x</td> | <td>1x</td> | ||
</tr> | </tr> | ||
- | <tr><td> | + | <tr><td>Vector</td> |
- | <td> | + | <td>1μl</td> |
- | <td> | + | <td>20-100μg</td> |
</tr> | </tr> | ||
- | <tr><td> | + | <tr><td>Insert</td> |
- | <td>0. | + | <td>1μl</td> |
- | <td></td> | + | <td>1:1 molar ratio</td> |
+ | </tr> | ||
+ | <tr><td>T4 DNA Ligase</td> | ||
+ | <td>0.2μl</td> | ||
+ | <td>5U/μl</td> | ||
</tr> | </tr> | ||
</table> | </table> |
Revision as of 10:05, 26 July 2013
Ligation
Materials:
- H2O
- 1.5ml tubes
- Ligation buffer
- Ligase
- Insert
- Vector
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 | 0.2μl | 5U/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 1h at 37°C.
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf