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Team:Newcastle/Ligation
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| - | For sticky end ligations incubate on the bench of 3-4 | + | For sticky end ligations incubate on the bench of 3-4 hours or overnight. |
| + | Important point to note is that 1:3 dilution means that during ligation, for 1 unit of vector, there is 3 units of insert and for 1:5 ligation, for 1 unit of vector there is 5 units of insert. | ||
==Notes== | ==Notes== | ||
Revision as of 08:50, 30 July 2010
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Ligation
Procedures
Set up the ligation mix according:
| Reagents | 1:3(μl) | 1:5(μl) | Vector(μl) |
|---|---|---|---|
| Vector | 1 | 1 | 1 |
| Insert | 3 | 5 | N/A |
| 10X BUFFER | 1 | 1 | 1 |
| T4 Ligase | 1 | 1 | 1 |
| H2O | 4 | 2 | 7 |
| Total Volume | 10.0 | 10.0 | 10.0 |
For sticky end ligations incubate on the bench of 3-4 hours or overnight. Important point to note is that 1:3 dilution means that during ligation, for 1 unit of vector, there is 3 units of insert and for 1:5 ligation, for 1 unit of vector there is 5 units of insert.
Notes
- For standard ligation 100-200ng of vector DNA is required - can be checked using nanodrop
- A ligation calcultion can be used however a 1:3 and a 1:5 ratio tends to be used instead.
   
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