Table 3. Comparison of the classical and the ligation-independent expression cloning workflows.

Classical approach using restriction enzymes and picking ofindividual E. coli colonies	Ligation-independent approach avoiding the use of restrictionenzymes and clone picking
Generate and purify PCR fragment	Generate and purify PCR fragment
Prepare vector for ligation	Prepare vector for annealing
Restriction enzyme treatment	T4-DNA polymerase treatment
Purify DNA insert	–
Ligation of fragment with prepared vector	Annealing of fragment with prepared vector
Transformation into competent bacteria	Transformation into competent bacteria
Plate and grow on solid media	Grow in bulk in liquid culture
Pick individual colonies (clones)	–
Grow clones in liquid culture	–
Isolate plasmid DNA from colonies	–
Analyze plasmid DNA from colonies	–
Grow correct clone in liquid culture	–
Isolate plasmid DNA	Isolate plasmid DNA
Transfect eukaryotic cells with expression plasmid	Transfect eukaryotic cells with expression plasmid