IGEM:Peking/2007/Count:Ligation
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- Takara T4 DNA ligase
- Choose reaction volume: 10-20 μL
- Mix proper proportion (usually 1:1) of DNA fragment and vector.
- Add 10×(meaning 1/10 of final volume) ligase buffer.
- Add 1 μL ligase per 10 μL final volume.
- Bath overnight in 16℃ water, or longer in 4℃.
- Begin transformation.
- New England Biolabs T4 DNA ligase
- Choose reaction volume: 10-20 μL
- Mix proper proportion (usually 1:1) of DNA fragment and vector.
- Add 10×(meaning 1/10 of final volume) ligase buffer.
- Add 0.5 μL ligase per 10 μL final volume.
- Bath in 16℃ water for 10 min, or in room temperature for convenience.
- Deactivation: 65℃ water for 10 min (longer ligation time or skipping deactivation step may lead to high pseudo-positive ratio.
- Begin transformation.