Ligation
Protocol
- Determine the amount of plasmid and insert to be used through the equation X ng plasmid/[(1 M) X bp] = X ng insert/[(X M) X bp]. It is best to have a pasmid to insert ratio between 1:4 and 1:10. Note: a useful website for this calculation is http://www.insilico.uni-duesseldorf.de/Lig_Input.html. Also, using 150 ng of vector is a good starting point.
- Add the following volumes to a round-lidded PCR tube:
| Reagent |
Volume (μL) |
|---|---|
| DNA |
X of insert X of plasmid |
| 5x T4 DNA ligase buffer |
4 |
| T4 DNA ligase |
1 |
| ddH2O |
up to 20 μL |
| Total |
20 |
Note: Larger total volumes can be used; however, it is best to keep the total volume as small as possible, without going below 20 μL.
- Gently mix by tapping the side of the tube.
- Centrifuge the tube for 5 seconds at 4,500 xg
- Leave tube at room temperature overnight.