Team:Arizona State/Lab/Protocols/Ligation

DNA Ligation Protocol From OpenWetWare

50 uL Ligation:

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