IGEM:Peking/2007/Count:Ligation

• Takara T4 DNA ligase

1. Choose reaction volume: 10-20 μL
2. Mix proper proportion (usually 1:1) of DNA fragment and vector.
3. Add 10×(meaning 1/10 of final volume) ligase buffer.
4. Add 1 μL ligase per 10 μL final volume.
5. Bath overnight in 16℃ water, or longer in 4℃.
6. Begin transformation.

• New England Biolabs T4 DNA ligase

1. Choose reaction volume: 10-20 μL
2. Mix proper proportion (usually 1:1) of DNA fragment and vector.
3. Add 10×(meaning 1/10 of final volume) ligase buffer.
4. Add 0.5 μL ligase per 10 μL final volume.
5. Bath in 16℃ water for 10 min, or in room temperature for convenience.
6. Deactivation: 65℃ water for 10 min (longer ligation time or skipping deactivation step may lead to high pseudo-positive ratio.
7. Begin transformation.