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BISC110: Series 4 Lab 11 Enzymology: Difference between revisions
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Before you begin your experiment, you need to get organized. Enzyme assays require precise timing since enzymes are not consumed by the reactions they catalyze. Consequently, enzymes will continue to drive a reaction until the reaction has reached equilibrium. Instead of waiting for equilibrium, you will perform your assays so that each test tube will react for exactly 15min. This means you will add sucrose to the invertase that is in the test tube, staggering the time you start each reaction. Precisely 15min after you have started each invertase catalyzed reaction, you will end it by adding the DNS reagent. The DNS reagent raises the pH in the reaction tube and inactivates the invertase. | Before you begin your experiment, you need to get organized. Enzyme assays require precise timing since enzymes are not consumed by the reactions they catalyze. Consequently, enzymes will continue to drive a reaction until the reaction has reached equilibrium. Instead of waiting for equilibrium, you will perform your assays so that each test tube will react for exactly 15min. This means you will add sucrose to the invertase that is in the test tube, staggering the time you start each reaction. Precisely 15min after you have started each invertase catalyzed reaction, you will end it by adding the DNS reagent. The DNS reagent raises the pH in the reaction tube and inactivates the invertase. | ||
'''Procedure for Invertase Assays''' | =='''Procedure for Invertase Assays'''== | ||
1. Obtain 8 x 13mm test tubes, and label them 1–8 with a Sharpie® permanent marker. | 1. Obtain 8 x 13mm test tubes, and label them 1–8 with a Sharpie® permanent marker. | ||
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17. Wipe the outside of each test tube with a Kimwipe™ or if a film still remains on the tube, transfer the contents to a new 13mm test tube. Using tube #1 to blank the spectrophotometer, read the absorbance of each reaction at 540nm and record all data in your lab notebook. Were the activity levels of the invertase produced by the yeast cells higher or lower than the activity of the purified invertase? | 17. Wipe the outside of each test tube with a Kimwipe™ or if a film still remains on the tube, transfer the contents to a new 13mm test tube. Using tube #1 to blank the spectrophotometer, read the absorbance of each reaction at 540nm and record all data in your lab notebook. Were the activity levels of the invertase produced by the yeast cells higher or lower than the activity of the purified invertase? | ||
'''Laboratory Clean up''' | ''' Laboratory Clean up ''' | ||
<br> | |||
Pour the DNS solutions in the waste jar in the hood. You may want to wear gloves to avoid yellow stains on your hands. <br> | Pour the DNS solutions in the waste jar in the hood. You may want to wear gloves to avoid yellow stains on your hands. <br> | ||
Discard the empty test tubes from the invertase assay in the glass recycle container at the front of the lab. <br> | Discard the empty test tubes from the invertase assay in the glass recycle container at the front of the lab. <br> | ||
