Molecular Methods Outline

Molecular Methods Outline - MCB121 C S Gasser Fundamentals...

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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 Reading: Lecture handout from Smartsite web. MBOG Ch.21 pp. 746-750, 751. Problems : 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 I. Cloning – Biological replication of genetically identical entities. Examples: colonies of microbes, cloned animals, vegetative propagation of plants, molecular cloning. II. Basic Tools A. Restriction endonucleases, ligase B. Plasmids e. g. pBluescript II - origins, selectable marker, scoreable marker, insertion site C. Making a clone Forced cloning (asymmetric) Symmetrical cloning III. Additional Plasmid Vector Features A. Phage promoters B. Expression vectors IV. Vectors for larger inserts A. Lambda vectors - 25 kb inserts (total of lambda plus insert DNA must be 42 - 52 kb for replication and packaging). B. 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). C. YACs – Yeast artificial chromosomes. Include yeast origin of replication (ARS), centromeres (CEN) and Telomeres. 100 – 1,000 kb inserts. V. Libraries 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)
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Cross Index of Recognition Sequences Palindromic Tetra- and Hexa-Nucleotide Recognition Sequences Sequences at the top of each column are written 5´ to 3´ according to convention. Open squares at the left of each row are place holders for nucleotides within a restriction endonuclease recognition sequence; arrowheads indicate the point of cleavage. Sequences of complementary strands and their cleavage sites are implied. Enzymes written in blue type recognize only one sequence while enzymes written in black type recognize multiple sequences. BsaW I BsaA I Ban I A T A T A T A T A T C G C G C G C G C G G C G C G C G C G C T A T A T A T A T A AATT ACGT AGCT ATAT CATG Tsp509 I CCGG CGCG CTAG GATC GCGC GGCC GTAC TATA TCGA TGCA TTAA Dpn II Msp I Alu I Bst U I Mbo I Sau3A I Bfa I
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Molecular Methods Outline - MCB121 C S Gasser Fundamentals...

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