Question

Why are two different restriction enzymes used to cut the puc19 plasmid and the lux gene DNA? What would have happened if only the HinD III enzyme was used?

Answer 1

This is because there might not be one restriction site bordering the gene to be cloned and the identical restriction site in the plasmid. Since these two restriction enzymes make compatible sticky ends, the insert has a chance of combining with the plasmid. To ensure efficient digestion, the two recognition sites should be more than 10 base pairs apart. If one of the enzymes is a poor cutter or if the sites are separated 10 base pairs or less, the digestions should be performed sequentially