IGEM:Peking/2007/Count:Ligation

From OpenWetWare

Jump to: navigation, search
  • 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.
Personal tools