Ligation

Ligation Protocol with T4 DNA Ligase

1. Set up the following reaction in a microcentrifuge tube on ice.
   1. 2uL of 10X T4 DNA ligase Buffer
   2. 50 ng 50 ng (0.025 pmol) vector DNA (3 kb)
   3. 50 ng (0.076 pmol) insert DNA (1 kb)
   4. 1 uL of T4 DNA Ligase
   5. add nuclease-free water to make the total volume 20 uL
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours(alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).

N.B (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.)

troubleshooting

1. to avoid frequent thawing and freezing of the ligase buffer, prepare working stock of ligation buffer