L4 - Lab 4 - Bacteria, Complementation, & Photosynthesis

L4 - Lab 4 - Bacteria, Complementation, &...

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Office Hours (2084 VLSB) Mikes: M 11-1, W 2-3. 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. Genetics (GMB II) is difficult. Prepare in advance for the genetics-- will tie in genetics within the complementation experiments.
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Culturing bacteria, Complementation and Photosynthesis Readings: Lab Manual pgs 60-86 (73-74), 107-114 (107-109), 115-129 Campbell Ch. 10
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Standard Curves: Application Part I Enzyme lab - vary enzyme concentration 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 50 100 150 200 250 300 350 400 450 nanogram/ml amylase Optical Density (O.D. units) Lab 3: Amylase activity in saliva (Part IIC) i) activity of diluted saliva about 160 ng about 40 ng ii) conversion factor of dilution if 1,000 fold then 160 μ g if 10,000 fold then 400 μ g mean value 280 μ g
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Overview: Isolation Restriction Digestion Ligation Transformation Screening
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Ligate Digest Transform & screen
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Purified ! DNA A AGCTT---A AGCTT TTCGA A---TTCGA A Ligation reaction in lab 2 R.E. R.E. Isolate & Digest Anneal & Ligate Transform & screen Amp R X-gal to Blue When ?
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Isolation and Culturing Transformed E. coli . Incubate 37 o C overnight Media = LB/AMP GMB I Plasmid isolation DNA digestion GMB II Electrophoresis Controls: + (use known pBLU) and - (No DNA) LB LB/AMP/X-gal LB LB/AMP/X-gal Blue loop
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Complementation: Key concepts i) Ploidy -sets of chromosomes
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Loss of function Mutants: single mutation, recessive Info from Mom Info from Dad
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L4 - Lab 4 - Bacteria, Complementation, &...

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