"
Team:EPF-Lausanne/Protocols/Ligation
From 2009.igem.org
(Difference between revisions)
(→Reaction mix) |
|||
| (9 intermediate revisions not shown) | |||
| Line 15: | Line 15: | ||
<html><center> | <html><center> | ||
| - | <font size="6" color="#007CBC" | + | <font size="6" color="#007CBC">Ligation Protocol</font> |
</center></html> | </center></html> | ||
<br> | <br> | ||
| - | + | ||
| - | + | ||
| - | + | ||
==Reaction mix== | ==Reaction mix== | ||
| - | {| class="wikitable" style="text-align: | + | {| class="wikitable" style="text-align:left; width:20%;" |
|- | |- | ||
! scope=row | T4 ligase buffer | ! scope=row | T4 ligase buffer | ||
| Line 34: | Line 32: | ||
|} | |} | ||
| - | + | 6:1 molar ratio of insert to vector (~10 ng of vector). | |
| - | 6:1 molar ratio of insert to vector (~10 ng of vector) | + | <br>Complete with dH2O up to 10 µl. |
| - | Complete with dH2O up to 10 µl | + | |
==Molar ratio== | ==Molar ratio== | ||
| - | + | [[Image:Formule_insert.jpg|center|formule]] | |
| - | + | ||
==Notes== | ==Notes== | ||
| - | In general, we can take 1 µl of vector for 3 µl of insert. | + | In general, we can take 1 µl of vector for 3 µl of insert. But you can change the molar ratio in case the sizes of the insert and the vector are really different. |
| - | + | ||
| - | + | ||
| + | To control ligation specificity, you need to have a '''control with the vector only'''. | ||
==Incubation== | ==Incubation== | ||
| - | + | 1h at Room Temperature. | |
</div><div CLASS="epfl09bouchon"></div> | </div><div CLASS="epfl09bouchon"></div> | ||
Latest revision as of 18:45, 20 October 2009
Contents |
Reaction mix
| T4 ligase buffer | 1 µl |
|---|---|
| T4 ligase | 0.5 µl |
6:1 molar ratio of insert to vector (~10 ng of vector).
Complete with dH2O up to 10 µl.
Molar ratio
Notes
In general, we can take 1 µl of vector for 3 µl of insert. But you can change the molar ratio in case the sizes of the insert and the vector are really different.
To control ligation specificity, you need to have a control with the vector only.
Incubation
1h at Room Temperature.
