L5 - Lab 5 - GMB I - Plasmid Isolation and restriction...

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Plasmid Isolation and restriction digestions, Complementation and GMB I (Mitosis & Meiosis & DNA sequencing) Readings: Lab Manual pgs 60-86 (75-80), 107-114 (110-112), 131-140. Campbell Ch. 12-17, 20, pages, 7 th ed. 346-350 or 8 th ed. 561-564
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Office Hours (2084 VLSB) Mikes: M 11-1, W 1-2. Lab GSIs: M 2-4, T 8: 30 -9: 30 , 1-2, 5: 30 -6: 30 , W 1-2, 5: 30 -6: 30 Th 8: 30 -9: 30 , 1-2, 5: 30 -6: 30 , F 1-2. Disc. GSIs: M, T, F 10-2 W 11-2, Th 10-12 Study resources - webcasts, archived webcasts, reviews (4 hours), office hours. Review Friday Genetics (GMB II) is difficult. Prepare in advance for the genetics-- will tie in genetics within the complementation experiments.
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Time O.D. i) Blank ii) Dark iii) Light + DCMU iv) Light v) Light + CH3NH2 Controls: light vs. dark
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6. What results would you predict if both DCMU and methylamine had been added at the same time? Explain. 7. How much NADP + is being reduced, relative to DCPIP, in the light under your conditions? Less, about the same, or more? Explain.
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GMB I lecture outline • Overview of cloning • Plasmid isolation • DNA digestion • DNA sequencing • Complementation • Human life cycle- mitosis and meiosis
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Ligate Digest Transform & screen- involves plasmid isolation, digestion and electrophoresis.
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Annealing & ligation Amp R Reform restriction sites Transformation Isolate plasmid DNA Isolate source DNA R.E. digestion R.E. digestion Plasmid Isolation R.E. digestion Gel Electrophoresis Data analysis (size)
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Plasmid Isolation Key points 1) Ampicillin S vs. R 3) Bacterial chromosome vs. plasmid DNA 5) Cell fractionation/centrifugation 4) Binding affinities
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Lysis releases components. Role of NaOH, SDS.
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This note was uploaded on 09/28/2009 for the course BIO 1al taught by Professor Pederson during the Fall '08 term at University of California, Berkeley.

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L5 - Lab 5 - GMB I - Plasmid Isolation and restriction...

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