MCB121, C. S. Gasser
Fundamentals of Recombinant DNA, C. Gasser 1/29/2008
Dr. Gasser's office hours
- Wed. 10 - 11, 2061 SLB, Fri. 1:30 – 2:30 3061 SLB
Lecture handout from Smartsite web. MBOG Ch.21 pp. 746-750, 751.
MBOC 8-52 – 8-62 (excluding 8-59), 8-69, 8-74,
8-76, 8-80, 8-81, 8-87, 8-
91; Molecular Methods Problem Set on lecture Web Resources page on Smartsite
– Biological replication of genetically identical entities. Examples: colonies
of microbes, cloned animals, vegetative propagation of plants, molecular
II. Basic Tools
A. Restriction endonucleases, ligase
e. g. pBluescript II - origins, selectable marker, scoreable marker, insertion site
C. Making a clone
Forced cloning (asymmetric)
III. Additional Plasmid Vector Features
A. Phage promoters
B. Expression vectors
IV. Vectors for larger inserts
Lambda vectors -
25 kb inserts (total of lambda plus insert DNA must be 42 -
52 kb for replication and packaging).
BACs – Bacterial artificial chromosomes. Include the origin of replication from
the F plasmid. 70 – 300 kb inserts (usually ~100 kb, but can be up to 1,000 kb).
YACs – Yeast artificial chromosomes. Include yeast origin of replication (ARS),
centromeres (CEN) and Telomeres. 100 – 1,000 kb inserts.
A. Genomic Libraries
B. cDNA Libraries
VI. Library screening
A. Plaque/colony lifts and nucleic acid hybridization.
B. Antibody screening of expression libraries.
VII. Polymerase Chain Reaction (PCR)