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Team:Newcastle/Ligation
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|3 | |3 | ||
|5 | |5 | ||
| - | | | + | |N/A |
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|'''10X BUFFER''' | |'''10X BUFFER''' | ||
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| - | For sticky end | + | For sticky end ligation, incubate the tube containing above mentioned chemicals on the bench for a period of 3-4 hours or overnight. |
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| + | 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== | ||
| - | * For standard ligation 100- | + | * For standard ligation, 100-200 ng of vector DNA is required and this can be checked using nanodrop protocol (refer to [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] protocol). |
| + | |||
| + | *Ligation calculation can be used to calculate the amount of vector and insert required for the ligation but 1:3 or 1:5 ligation ratios are used more frequently. | ||
| - | |||
| + | '''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' | ||
{{Team:Newcastle/footer}} | {{Team:Newcastle/footer}} | ||
Latest revision as of 14:37, 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 ligation, incubate the tube containing above mentioned chemicals on the bench for a period 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, 100-200 ng of vector DNA is required and this can be checked using nanodrop protocol (refer to Nanodrop Spectrophotometer protocol).
- Ligation calculation can be used to calculate the amount of vector and insert required for the ligation but 1:3 or 1:5 ligation ratios are used more frequently.
Go back to our Protocol List
   
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