BIOS 2011 Lab 1 Enzymes

The time it takes to reach this lack of reaction with

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Unformatted text preview: he amylose forms a tight helix around I5 ­ ions (a string of five iodine atoms with a minus one charge), which turns the test solution opaque blue ­black. During each trial of a ­Amylase digesting starch you will pipette off small amounts of liquid from the reaction test tube to a spot plate with IKI, to see if starch is still present. As the starch begins to disappear, later tests will no longer change blue ­black with the indicator but will be dark brown, then a lighter red ­brown, showing the digestion of the starch has progressed to smaller dextrins, shorter polymers of glucose. At the end of the reaction, the test results will show no reaction with the indicator, which will remain a light yellow. The time it takes to reach this lack of reaction with the indicator, when all the starch has been hydrolyzed to maltose and maltotriose, is the endpoint time you will record. You will then calculate rate by dividing the amount of starch hydrolyzed by the endpoint time. 5 Exercise 1 Enzymes PROTOCOL 1: THE INHIBITION OF SUCCINIC DEHYDROGENASE BY MALONIC ACID 1. Label 6 small test tubes with numbers 1 ­6 using a Sharpie or other lab marker. 2. Refer to the table below for the volumes of reagents to be added to each tube. Using micropipetters add the all the listed reagents to the 6 tubes, except the Calf heart. Calf heart should be added last. Tube Succinic Acid Methylen e Blue Deionize d H2O Other Calf Heart 1 90 µL 210 µL 270 µL 500 µL Sucrose 0 µL 2 90 µL 0 µL 480 µL — 500 µL 3 0 µL 210 µL 360 µL — 500 µL 4 90 µL 210 µL 270 µL — 500 µL 5 90 µL 210 µL 240 µL 30 µL Malonic acid 500 µL 6 330 µL 210 µL 0 µL 30 µL Malonic acid 500 µL 3. Add the Calf heart to the tubes and mix vigorously by Pasteur pipette, or use a vortex mixer if you prefer. Note that Tube 1 has no Calf heart, but uses plain Sucrose instead. If you have measured all reagents and the deionized water correctly the tubes should all have the same total volume of liquid, and the menisci should be at the same height in all tubes. 4. Patiently and slowly pipette 250µl mineral oil into each tube to form a continuous layer 1 ­2 mm (or c. 1/16”) thick on the surface above the reactants. Mineral oil is very viscous and must be pipetted more slowly than aqueous solutions. This will keep oxygen from entering the reaction to re ­oxidize any Methylene White. 5. Make a note of the initial color in each tube in the worksheet below. Place your rack of small test tubes into the 37°C water bath nearest your bench. Incubate for 1 hou...
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This note was uploaded on 02/09/2012 for the course BIOLOGY 102 taught by Professor Anderson during the Spring '11 term at Harvard.

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