50 uL Ligation (from [http://openwetware.org/wiki/DNA_Ligation OpenWetWare]):
- Add 22.5 uL deionized H20 to sterile eppendorf tube
- Add 5 uL of ligation buffer to the tube
- Vortexing buffer before pipetting helps ensure that it is well mixed
- Add 15 uL of insert to the tube
- Add 5 uL of vector to the tube
- 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)
- Let 50 uL solution sit at 22.5 C (room temperature) for at least 30 mins
- Heat inactivate ligase at 65 C for 10 mins
- Store at -20 C or proceed to transformation
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