week_2_lecture_1 - Required readings for week 2 WEAVER...

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Required readings for week 2 WEAVER, MOLECULAR BIOLOGY. 5th Ed. Chapter 4, pp. 49-69, Chapter 5, pp. 75-79, 82-83, 85-97, Chapter 24, pp. 769-770
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Lecture 1 (week 2) Recap from week 1 1st hour: quiz and discussion (15 min)  DNA isolation, restriction enzymes, agarose gel  electrophoresis, restriction mapping Break 2nd hour: DNA hybridization, DNA fingerprinting,  DNA cloning/vectors, replica platting
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RECAP FROM PREVIOUS LECTURE - THE DNA DOUBLE HELIX 1. Clues: Griffith/Avery transforming principle & Hershey /Chase; Chargaff Rule; X = helix; Nucleotide chemistry 2. Implications of the double helix: Tm, semi-conservative replication THE TRIPLET CODE 1. in vitro : Nirenberg and Khorana – tRNA/mRNA binding assays 2. in vivo : Crick and Brenner – rII phage T4 3. 43 = 64 codons - 61 code for amino acids, 3 code for translation stop codons DNA TOPOLOGICAL STRUCTURE 1. A, B, and Z-forms of DNA 2. DNA supercoiling: DNA in vivo is “closed” and often supercoiled; topology: -ve supercoils (underwound = stored energy for rep or transcription); +ve supercoils (overwound) Topoisomerases: type I and II L = T + W where: L is linking no. (always an integer), T is twist (number of turns), and W is the writhe (no. of times the helix crosses over itself)
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DNA ISOLATION DNA genes are not biochemically distinct, i.e. unrelated similar-sized DNA molecules behave the same DNA purification: 1. Lyse cells 2. Remove debris after centrifugation 3. Separate DNA from protein (e.g. phenol extraction & CsCl gradient centrifugation) 4. Concentrate by ethanol precipitation and centrifugation
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RECOMBINANT DNA METHODS Recombinant DNA: joining different DNAs 1.
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