announcements post lab 3 and pre lab 4 are due by the time your lab meets next. lna enzymes is...
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AnnouncementsAnnouncements
Post Lab 3 and Pre Lab 4 are due by the time your lab meets next.
LNA Enzymes is assigned today, and due next week within the first 5 minutes of your lab period.
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Exercise 2B:Exercise 2B:
1. Experimental Plates: Examine your plates and observe the type of bacteria or fungal growth that appears on each.
2. Streak Plates: Examine the colonies; TA will be around to assign your “Skills” score
3. Complete your LNA: Exercise 2 and turn it in as directed by your TA.
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Exercise 3A: Exercise 3A: SpectrophotometrySpectrophotometry
Goals: Understand the process by which spectrophotometry
can be used to quantify experimental results. Develop skills taking measurements using a
spectrophotometer.
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SpectrophotometrySpectrophotometry
Measurement of light absorption or transmission through a solution
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Types of photometers:Types of photometers:
Colorimeters and spectrophotometers measure the amount of light absorbed by solutions.
Turbidimeters and nephelometers measure the light scattered by suspensions.
Fluorimeters measure the fluorescence produced by absorbed light.
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Light through a solution:Light through a solution:
Example ONP (o-nitrophenol) Absorbs blue light and allows yellow light to pass
through. Solution therefore appears yellow.
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Absorption SpectrumAbsorption Spectrum
A plot of the relative amount of light absorbed by a compound as a function of the wavelength
Absorption spectrum of ONP
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Components of a Typical SpectrophotometerComponents of a Typical Spectrophotometer
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3A Techniques:3A Techniques:
Using colored water, take measurements using a spectrophotometer.
Keep cuvettes free from fingerprints.
Align cuvette correctly each time you take a measurement.
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3B: Enzymes3B: Enzymes
Goals: Describe the principles of enzymatic reactions Use the principes of spectrophotometry to determine
the concentration of the product of an enzyme-catalyzed reaction.
Determine the effect of ß-galactosidase concentration on the rate of cleavage of ONPG.
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Introduction:Introduction:
Enzymes increase the rate of reactions, but do not allow reactions to occur that could not occur otherwise.
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There are two ways to increase the rate of There are two ways to increase the rate of a chemical reactiona chemical reaction
1. Increase the average kinetic energy by raising the temperature, or
2. Lower the activation energy by adding a catalyst
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Catalyzed Reaction Reach a Maximum Rate
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Enzyme RegulationEnzyme Regulation
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-galactosidase-galactosidase
O-nitrophenyl--D-galactopyranoside (ONPG)
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Hypothesis GenerationHypothesis Generation
Identify one characteristic you expect to change as you add ONPG, buffer, and enzyme
A yellow solution is produced as o- nitrophenolate is produced
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Rephrase your speculation to the if, then format
If ONPG is catalyzed by -galactosidase, and I add the enzyme in various amounts, the products of o-nitrophenolate (yellow color) will differ as well
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Independent VariableIndependent Variable
Amount of -galactosidase
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Dependent VariableDependent Variable
OD420 reading of o-nitrophenolate produced
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ControlsControls
ONPG + Buffer, but no enzyme added
and
Buffer + enzyme, but no ONPG added
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The Experiment:The Experiment:
Take measurements of various amounts of ONPG catalyzed by -galactosidase