Ligation Protocol

Ligation Protocol with T4 DNA Ligase

1.     Set up the following reaction in a 1.5 mL Eppendorf tube on ice '''adding the T4 DNA Ligase last'''. 2.     Gently mix the reaction by pipetting up and down and microcentrifuge briefly.

3.     For sticky ends, Incubate at room temperature for 10 minutes.

4.     For blunt ends, Incubate at room temperature for 2 hours.

5.     Chill on ice and transform the total volume of the reaction into 50 uL of competent cells.

Calculation for Insert amount: Convert ng amounts to uL based on concentration of digestions.

Ratio = amount of insert to amount of vector. For our ligations, we usually use a 3:1 ratio.