IGEM:PennState/2006/Ligation
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Ligation
Time: 15 min
Reference: NEB T4 ligase technical bulletin http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0202.pdf
Pre
- Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio.
Ligation
- for 20 μL reaction volume, to eppendorf add:
| Stuff | Volume (uL) |
|---|---|
| insert | x |
| vector | y |
| dH2O | z |
| 10X T4 ligase buffer | 2 |
| T4 ligase | 0.5 |
| Total | 20 |
where x is usually 5-15 uL, y=[1,5] uL.
Also, always remember to do a negative control, which will be like the reaction above, but w/ no insert. Your transformation plates will then be an measure of any vector re-ligation/uncut background.
2. For overnight ligation, add ice to cold water in styrofoam container to bring temp to 12-14ºC. Incubate overnight (15 hr.). Alternatively, for quicker ligation, ligate at RT for 30 min. Proceed directly to transformation.
