In a cloning workflow, digestion with BamHI and HindIII is used to do what?

In a cloning workflow, digestion with BamHI and HindIII is used to do what?

• A. Change plasmid antibiotic resistance
• B. Increase plasmid copy number
• C. Inactivate restriction enzymes
• D. Create compatible ends for ligation into a vector

Answer: (D)

In cloning workflows, restriction digestion is used to create ends that can be joined to a vector. When both the plasmid and the DNA fragment are cut with BamHI and HindIII, you generate compatible sticky ends on both pieces. These ends are complementary to the ends produced on the vector by the same enzymes, so the insert can anneal to the vector and be sealed by DNA ligase. Using two different enzymes also provides directionality, so the fragment inserts in a defined orientation rather than in either direction. This is why the purpose is to create ends that are suitable for ligation into a vector. It doesn’t change antibiotic resistance or plasmid copy number, and digestion itself doesn’t inactivate the enzymes.

In cloning workflows, restriction digestion is used to create ends that can be joined to a vector. When both the plasmid and the DNA fragment are cut with BamHI and HindIII, you generate compatible sticky ends on both pieces. These ends are complementary to the ends produced on the vector by the same enzymes, so the insert can anneal to the vector and be sealed by DNA ligase. Using two different enzymes also provides directionality, so the fragment inserts in a defined orientation rather than in either direction. This is why the purpose is to create ends that are suitable for ligation into a vector. It doesn’t change antibiotic resistance or plasmid copy number, and digestion itself doesn’t inactivate the enzymes.