- Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio (660 g/mole/base pair).
- for 20 μL reaction volume, to eppendorf add:
|10X T4 ligase buffer||2|
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.
- For overnight ligation, store at 16C (15 hr)