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Unformatted text preview: Lecture 3 S. Harmer Molecular Methods (Databases, hybridization, cloning, libraries, PCR) MCB 161 01/12/2010 Reading: Watson text: pp. 113 – 117; 739 - 752 Problems: MBOC Problems book: 4-55, 8-67, 8-80 (note there is a typo in the book; you should be able to derive the correct answer nonetheless), 8-87, 8-95; also, see problems on the web site. I. Databases Genbank, administered by the National Center for Biotechnology Information (NCBI) Database searching often done using the Basic Local Alignment Search Tool (BLAST), which can compare a given nucleic acid or protein sequence against all known genes C. E value (expect value) indicates significance of match; the lower the score, the more likely the match is to be significant A. B. II. Nucleic acid hybridization A. Melting temperature (Tm) can be estimated: Tm (°C) = 81 + 16.6 (log10 ci) + 0.4 [%(G+C)] - 0.6 (% formamide) -1.5(%mismatch) - 600/n (ci = cation concentration in M; n = length of DNA in base pairs) B. Hybridization C. Applications: Southerns, Northerns, microarrays III. Cloning A. What is cloning? B. Tools: restriction enzymes C. Tools: DNA ligase D. Tools: bacterial plasmids (vectors) need origin, selectable marker, cloning site(s); E. cloning techniques (symmetrical, directional) IV. Vector bells and whistles A. Expression vectors B. in vitro transcription and translation V. Libraries A. Genomic libraries B. Screening libraries with nucleic acid probes PCR: Review on your own V I. Lecture 3 S. Harmer Index of restriction enzyme sequences www.neb.com MCB 161 01/12/2010 Cartoon of Bluescript plasmid www.stratagene.com Portion of pMAL expression vector www.neb.com In vitro purification scheme www.neb.com ...
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- Spring '09