Team:Arizona State/Lab/Protocols/Ligation

From 2011.igem.org


Protocols: Ligation


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50 uL Ligation (from OpenWetWare):


  1. Add 22.5 uL deionized H20 to sterile eppendorf tube
  2. Add 5 uL of ligation buffer to the tube
    Vortexing buffer before pipetting helps ensure that it is well mixed
  3. Add 15 uL of insert to the tube
  4. Add 5 uL of vector to the tube
  5. Add 2.5 uL of ligase
    Pipetting up and down before adding to tube helps ensure that it is well-mixed
    (vortexing the ligase may be inappropriate due to the sensitivity of the enzyme)
  6. Let 50 uL solution sit at 22.5 C (room temperature) for at least 30 mins
  7. Heat inactivate ligase at 65 C for 10 mins
  8. Store at -20 C or proceed to transformation