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BIOS 2011 Lab 1 Enzymes

Label 4 large test tubes 1 25 5 10 with a marker these

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Unformatted text preview: r. You don’t need to record the reactions over time; you are only interested in the end result. 6. After the 1 hour incubation, gently remove tubes from the water bath, without shaking them any further. Record the changes from initial color. Discard liquid contents in sink, and disposable test tubes in glass waste box. Worksheet for your Succinic Dehydrogenase experiment results: You can record your observations here, but be sure to generate a new table for your lab writeup as described in Appendix I. 6 Exercise 1 Tube Enzymes Succinic Methylene Deionize Acid Blue d H2O Calf Heart Other 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 Notes: 7 Initial Color Final Color Exercise 1 Enzymes PROTOCOL 2: THE ROLE OF α  AMYLASE CONCENTRATION ON REACTION RATE 1. Label 4 Large test tubes 1%, 2.5%, 5%, 10% with a marker. These will be for making 4 stock concentrations of diluted α ­Amylase. Mix four tubes according to the table below. Use a micropipetter to measure the a ­Amylase and a 10mL glass pipette to measure the water. Vortex for 30 ­60 seconds to make sure the solutions are homogenous. Stock Conc. a Amylase Deionized H2O 1% 100 µL 9.9 mL 2.5% 250 µL 9.75 mL 5% 500 µL 9.5 mL 10% 1000 µL 9.0 mL 2. Label 4 Small test tubes for starch/buffer mixes as follows: B1, B2.5, B5, B10. These will have identical contents, but each will be used as the substrate to test a different percentage of α ­ Amylase. To each small tube add 2mL of starch and 2mL pH 7.0 buffer. Vortex 30 seconds. Important: Remember a reaction begins the moment the enzyme and substrate make contact, so run and time each reaction separately. One person should time the reaction and put IKI on the spot plate, the other should mix the a ­Amylase and starch and draw off drops to test with the indicator. 3. The lab partner doing the timing and indicator should put out 2 ­3 drops of IKI per well in a clean, dry spot plate. Put IKI in only a few wells at a time, as Iodine is fugitive and will volatilize away into the atmosphere. Put IKI in more wells later, as needed. 4. The other partner should start the reaction as follows: draw up 1000µL of the desired enzyme stock concentration into a micropipetter tip (this leaves 9mL in the large tube for redos and the subsequent pH experiments). Keep the micropipetter vertic...
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