5. Restriction enzymes Q & A In 1970, Hamilton Smith isolated HindIII (1st restriction enzyme well characterized and used for DNA cloning), which comes from Haemophilus influenzae . They are named based on genus & species of bacteria it was isolated from. (EcoRI = Escherichia coli , RY13). They cut DNA by cleaving phosphodiester bonds (in sugar-phosphate backbone) that join adjacent nucleotides Which was the first one well understood? How are they named? How do they work?

12. Vector Features Modern plasmid DNA cloning vectors usually consider 6 desirable features: 1. Size (must be small enough to separate easily) 2. Origin of replication (ori) - DNA sequence at which replication is initiated 3. Multiple cloning site (MCS) - a stretch of DNA with recognition sequences for common restriction enzymes (Engineered into plasmid so that digestion does not result in loss of DNA fragment)

17. Replica plating diagram

18. Vector Features 5. RNA polymerase promoter sequences - place where RNA polymerase binds to begin transcription 6. DNA sequencing primer sequences - known sequence that allows sequencing of cloned DNA fragments that have been inserted into the plasmid