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Bio 1AL Lect 2 - Bio 1A Lab Lecture Outline 2 Vibrio...

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Bio 1A Lab Lecture Outline # 2 Vibrio streaking & Enzymes February 1 st , 2010 I. Vibrio isolation. A. Plate sea water onto SWC plate (Lab 1) B. Pick bioluminescent colony and streak onto SWC plate (Lab 2) C. Pick bioluminescent colony and streak onto SWC plate (Lab 3) D. Pick bioluminescent colony and set up a PCR reaction (Lab 4) E. Analyze PCR reaction – clean up and run a gel (Lab 5) i) If successful set up sequencing reactions (forward and reverse) F. Data anlysis – consensus sequence, Blast, Clustal (Lab 6) II. Light Spectroscopy A. Overview B. Identification – Absorption Spectrum C. Quantification – (A = clZ) D. Blank – why? III. Protein Structure: A. Amino Acids: classified by R group side chains a. Hydrophobic b. Hydrophilic c. Ionizable: NH2, COOH B. Polymerization C. Levels of structural organization: a. 1 o - primary sequence b. 2 o – hydrogen bonds from backbone molecules c. 3 o - prosthetic groups in addition d. 4 o – requires > 1 subunit IV. Reactions: Substrate(s) Product(s). A. Progress of Reaction: Δ G o , Δ S ± , E a . B. Boltzman – Energy Distribution. a. Heating does not change Δ G o nor E a , but the energy distribution changes. V. Enzymes A. Enzymes speed up chemical reactions by decreasing E a . B. How do enzymes work? a. Local environment, pH, proper orientation, distortion, change strength of binding. C. Isoenzymes (Isozymes). VI Measuring Kinetics A. Substrate Product. B. Direct/Indirect Measurements.
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VII. SalivaryAmylase A. Starch (Amylose) ------------> Maltose. B. Structure of Starch – (polymer α 1,4 glucose), focus terminal glucose: cyclical linear. C. Conversion – what happens: [maltose] & [starch/amylose]. a. Measure maltose indirectly by number of reducing units. D. Measure – Reduction a. Maltose + DNS (yellow) -------> Soluble brown substance; measure O.D. 510 nm b. Glucose – linear (L) vs. ring (R) form. c. amylose – terminal glucose (L) to (R) A (R) --- B (R) --- C (R) -- D (R) --- E (R) ---F (R) A (R) - B (R) - C (R) - D (R) - E (R) -F (L) d. Example: Amylose (chain of 6 Glucose) 3 Maltose 1 reducing unit is converted into 3 reducing units Actual difference = 2 reducing units! We need a blank. VIII. Measuring Kinetics: Part I A. Δ [E] 50ng 400ng a. Excess Substrate. 1 ml of S b. Keep all parameters constant except one variable. B. Experimental Setup: Table on P. 60 a. Blank: 0.5 ml 100ng/ml amylase, 1 ml DNS, 1 ml S (mix stock prior to removing) b. Reaction tubes!
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