1. Recombinant DNA technology
Allison, Ch8: Recombinant DNA technology and molecular cloning

2. Historical perspective
Labmda phage

4. Restriction modification system

7. Restriction enzymes

9. DNA ligase

10. Sticky end ligation
Blunt end ligation
Modified blunt end ligation

11. Modified blunt end ligation

12. Molecular cloning

13. Vectors Carry DNA molecules Independent replication (origin)
Unique restriction sites (MCS)
Selectable marker
Easy to transfer and recover from host

14. Choice of vectors

15. Plasmid
Nomenclature
pBR322 (p-plasmid, BR – origin: Bolivar and Rodriquez, 322 identifier)
pUC18 (p-plasmid, UC- university of california, 18 – identifier)

16. Cloning using plasmid vector

20. Transformation and screening
Transformation (competent cells)
Recombinant selection
Blue-white screening

21. Constructing libraries
Library
Genomic library
cDNA library

22. Probes Homologous Heterologous EST probes
Labeling an detection methods
Radioactive
Enzyme conjugates
Chromogenic
Fluorescent

23. Radioactive labeling

24. Labeling methods

27. Library screening

30. Restriction mapping

31. Electrophoresis

32. RFLP

33. Southern hybridization

37. DNA sequencing

39. Automated sequencing