"
Team:Newcastle/Ligation
From 2010.igem.org
(Difference between revisions)
(→Ligation) |
Swoodhouse (Talk | contribs) m (TeamNewcastleLigation moved to Team:Newcastle/Ligation) |
||
| (8 intermediate revisions not shown) | |||
| Line 2: | Line 2: | ||
=Ligation= | =Ligation= | ||
| - | |||
| - | |||
| - | |||
| - | |||
==Procedures== | ==Procedures== | ||
| Line 12: | Line 8: | ||
{|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 | ||
| - | | | + | |N/A |
|- | |- | ||
| - | | | + | |'''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 | ||
| Line 48: | Line 44: | ||
|} | |} | ||
| - | 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. |
| + | |||
| + | 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 [[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
| |||||||||||||
| |||||||||||||
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
   
|
    
|
