T4 DNA Ligase Ⅱ

Storage Solution: 400U/μl T4 DNA Ligase、10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol (pH 7.4 @ 25°C) 10*Reaction Buffer: 500 mM Tris-HCl、100 mM MgCl2、10 mM ATP、100 mM DTT (pH 7.5 @ 25°C)

1. Set up the following reaction in a microcentrifuge tube on ice. Add the following components in sequence. Note that the table shows a ligation using a molar ratio of 1:5 vector to insert for the indicated DNA sizes.

2. Gently mix the reaction through the up and down pipette, and inhale the liquid briefly.

3. For sticky ends, incubate at 16°C overnight or at room temperature for 10 minutes.

4. For blunt ends or single base overhangs, incubate overnight at 16°C or room temperature for 2 hours (alternatively, high concentrations of T4DNA ligase can be used for 10 minutes of ligations).

5. Chill on ice and transform 1-5 μl of the reaction into competent cells.

1.ATP is an essential cofactor in the reaction. This is in contrast to E. coli DNA Ligase, which requires NAD as a cofactor.

2.If T4 DNA Ligase is to be diluted, it is recommended that it be diluted with 50% glycerol in storage buffer and stored at -20°C. 3. Room temperature ligation