LabPractical_REVIEW_069 - BIO 205 Review Exam covers (Lots...

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BIO 205 Review • Exam covers (Lots of stuff) – Making solutions – Microscopy – Population kinetics – Chromatography – Urchin and root embryonic development – Enzyme catalyzed reactions – Protein Extraction – Protein electrophoresis & Western Blotting – Plasmid Ligation and Bacterial Transformation – Isolation of Plasmid DNA – Analysis of Plasmid DNA – And more… Solutions 342.3 g mol × 1.50 mol 1000 mL soln. × 75 mL soln. = 38.5 g sucrose in 75 mL of solution Prepare 75 mL of a 1.5M sucrose stock solution: FW M conc. volume Solutions (1.50 M sucrose stock) v 1 = (1.25 M sucrose stock)(25 mL) Prepare 25 mL of a 1.25 M dilution from the stock solution. C 1 V 1 =C 2 V 2 V 1 = 20.8 m stock sucrose solution to make 25 mL of solution Serial dilution of salivary amylase 4m L L 4 mL Parallel dilution {C 1 V 1 =C 2 V 2 } stock C 1 C 2 V 2 V 1 Where? In vitro means within a test tube, or just outside a living organism (a cell). An example is In Vitro Fertilization. In vivo (Latin for (with)in the living) means within a living organism (a cell). For example, biochemical reactions can take place in vivo. In situ (a term used in biology) means to examine the phenomenon exactly in place where it occurs (without removing it in some special medium etc.). Usually means something intermediate between in vivo and in vitro.
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MICROSCOPY • Bright field • Phase contrast • Dark field • Fluorescence Some microscope stuff… • Magnification (ocular X objective) • Resolution(d ∝λ and 1/NA) • Abbe’s equation: d=0.5 λ /NA obj • Kohler resolution • Depth of field • Field of vision • Working distance • Ocular reticule • Stage micrometer W X Y Z
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X X Y Z
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Counting sperm • Dilution Factor (df) – If 300 cells counted and df is 200 – Then total number of cells is in undiluted solution? – =300 cells X 200 = 60,000 cells • Volume of grids in which sperm are counted – If 125 cells counted in volume of 1 X 10 -4 mL – Then total number of cells 125 cells /10 -4 mL – 1.25 x 10 6 cells/mL More counting • Sperm extract is diluted 400 times and there are 200 cells counted in a volume of 10 -4 mL . • What’s the total density of sperm per mL? =(200 cells) X ( 400 ) /10 -4 mL =8.0 X 10 8 cells/mL Natural log of the opical desnity of Vibrio natriegens in B-broth Medum at 35 O C y = 0.0335x - 2.5333 R 2 = 0.9954 -3 -2.5 -2 -1.5 -1 -0.5 0 0 50 100 150 200 Time (minutes) Growth kinetics • Slope = A/ t = k • k= specific growth constant (with unit of time) •T dbl = 0.693/k • lnA =lnA 0 + kt (y = b+mx) Strategy for isolating cellular organelles • 1. Select appropriate source of cells. • 2. Place intact cells in a protected environment. • 3. Break open cells. • 4. Separate organelle of interest from other components.
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(6.1-6.5) Isolation of chloroplasts and extraction and separation of pigments with TLC • Selecting spinach leaves • Placing cells in a protective environment (chloroplast isolation medium) • Breaking open cells (blender) • Separating organelles (chloroplasts) of interest, filtering & centrifugation • Homogenizing chloroplasts • Disrupting membranes • Separation of pigments with TLC
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LabPractical_REVIEW_069 - BIO 205 Review Exam covers (Lots...

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