Ligation Protocol for Cloning with Blunt/TA Ligase Master Mix (M0367)

Protocol

1. Transfer master mix to ice prior to reaction set up. Mix tube by finger flicking before use.


2. Combine 20–100 ng of vector* with a 3-fold molar excess of insert and adjust volume to 5 μl with dH₂O.


3. Add 5 μl of Blunt/TA Ligase Master Mix and mix thoroughly by pipetting up and down 7-10 times or by finger-flicking.


4. Incubate at room temperature (25°C) for 15 min, place on ice.


5. Use for transformation or store at -20°C.


6. Do not heat inactivate.
   
   Heat inactivation dramatically reduces transformation efficiency.
   
   * In-house testing has demonstrated that maximal transformation efficiency is achieved using between 20–100 ng of vector (blunt or sticky, including T-vectors) and a corresponding 3-fold molar excess of the insert to be ligated into the vector.