Ligation Reaction

Buffers

T4 DNA Ligase Reaction Buffer

Protocol

1. Resuspend DNA fragment(s) in 10 μl of TE Buffer.
2. Add 10 μl of kinase reaction.
3. Add 1.5 μl of 10X ligase buffer (500 mM Tris-HCl (pH 7.5), 100 mM MgCl₂, 100 mM DTT, 10 mM ATP).
4. Adjust to 23 μl with water.
5. Add T4 DNA Ligase 800 units (NEB).
6. Incubate at 16°C overnight.
7. Heat inactivate reaction 75°C for 5 minutes.