Ligation

Steps:

1. Add 2ul of digested plasmid backbone (25 ng)

2. Add equimolar amount of digested fragment (< 3 µl)

3. Add 1 ul 10X T4 DNA ligase buffer.

4. Add 0.5 ul T4 DNA ligase

5. Add water to 10 ul.

6. Ligation 16 °C over night.

7. Transform with 1-2 ul of product. In the case of low concentration of DNA ligated, purify and concentrate the ligation system.