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Team:Newcastle/Ligation
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{|border=1 | {|border=1 | ||
|- | |- | ||
| - | !Reagents | + | !'''Reagents''' |
| - | !1:3(μl) | + | !'''1:3(μl)''' |
| - | !1:5(μl) | + | !'''1:5(μl)''' |
| - | !Vector(μl) | + | !'''Vector(μl)''' |
|- | |- | ||
| - | | | + | |'''Vector''' |
|1 | |1 | ||
|1 | |1 | ||
|1 | |1 | ||
|- | |- | ||
| - | | | + | |'''Insert''' |
|3 | |3 | ||
|5 | |5 | ||
| | | | ||
|- | |- | ||
| - | | | + | |'''10X BUFFER''' |
|1 | |1 | ||
|1 | |1 | ||
|1 | |1 | ||
|- | |- | ||
| - | | | + | |'''T4 Ligase''' |
|1 | |1 | ||
|1 | |1 | ||
|1 | |1 | ||
|- | |- | ||
| - | |H2O | + | |'''H2O''' |
|4 | |4 | ||
|2 | |2 | ||
|7 | |7 | ||
|- | |- | ||
| - | |Total Volume | + | |'''Total Volume''' |
|10.0 | |10.0 | ||
|10.0 | |10.0 | ||
Revision as of 08:45, 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 | |
| 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 hrs or overnight
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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