"
Team:Groningen/protocols/Ligation
From 2013.igem.org
(Difference between revisions)
| Line 56: | Line 56: | ||
<tr> | <tr> | ||
<td>Vector</td> | <td>Vector</td> | ||
| - | <td>xµl</td> | + | <td ALIGN=CENTER>xµl</td> |
<td>20-100ng</td> | <td>20-100ng</td> | ||
</tr> | </tr> | ||
| Line 62: | Line 62: | ||
<tr> | <tr> | ||
<td>Insert</td> | <td>Insert</td> | ||
| - | <td>xµl</td> | + | <td ALIGN=CENTER>xµl</td> |
<td>3:1 molar ratio over vector</td> | <td>3:1 molar ratio over vector</td> | ||
</tr> | </tr> | ||
| Line 68: | Line 68: | ||
<tr> | <tr> | ||
<td>T4 DNA ligase 5U/µl</td> | <td>T4 DNA ligase 5U/µl</td> | ||
| - | <td>2µl</td> | + | <td ALIGN=CENTER>2µl</td> |
<td>0.5U/µl</td> | <td>0.5U/µl</td> | ||
</tr> | </tr> | ||
Revision as of 15:24, 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
iGEM 2013 Groningen
|
|
|
|
 |
|
