After DNA quantification with Nano-Drop, add to a 0.2-ml PCR tube (10 ul final volume):
- 20-40 ng of vector DNA
- ng of insert: 6 (insert length/vector length) (ng of vector DNA)
- NOTE: the factor 6 can be decreased up to a factor 2
- Milli-Q water up to 8 ul.
- 1 ul of Ligase Buffer (Roche)
- 1 ul of T4 Ligase (Roche)
Mix very well and incubate in the thermocycler (T4Lig program, reaction volume of 10 ul) at 16degC overnight.
Heat-inactivate the enzyme at 65degC for 10 min in the thermocycler (pretrasf program, reaction volume of 10 ul) before proceeding with transformation.