Team:Newcastle/Ligation
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For sticky end ligations incubate on the bench of 3-4 hours or overnight. | 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 | + | |
+ | Important point to note is that 1:3 dilution means that during ligation, for 1 unit of vector, there are 3 units of insert and in 1:5 ligation, for 1 unit of vector there is 5 units of insert. | ||
==Notes== | ==Notes== |
Revision as of 08:51, 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 are 3 units of insert and in 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.