BIOS 2011 Lab 1 Enzymes

10 seconds is good for 10 aamylase stock 20 sec for

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Unformatted text preview: al while it has liquid in the tip. 5. When the timer says “zero,” start timing, and simultaneously pipette the α ­Amylase rapidly into its starch/buffer mix test tube. Mix by pushing the plunger of the micropipetter up and down several times (10 ­15 quick pushes). Immediately draw up some of the reacting mix into the pipetter tip (the volume isn’t critical) and drop a few drops into the IKI in the first well of a spot plate. This initial spot should turn opaque blue/black. 8 Exercise 1 Enzymes 6. At regular intervals from the time zero, draw up a little more of the reacting mix from the test tube and add a few drops to the next well with fresh IKI. See if there is still a color change with the indicator. 7. Keep repeating step 6 until your drops from the reaction tube show no color change with the IKI. When these drops appear to be just diluting the yellow color of the indicator drops, that means the α ­Amylase has hydrolyzed all 10mg of starch substrate. Record the total time to endpoint on the worksheet below. You should not record times of intermediate colors, only the endpoint time. Clean spot plates with tap water and dry with a paper towel. Note: The drop test interval should be adjusted depending on the stock concentration of a ­Amylase being tested. 10 seconds is good for 10% a ­Amylase stock, 20 sec for 5%, 30 sec for 2.5% and 30 or 60sec for 1%. 8. Proceed to the next reaction for the next lower percentage stock of α ­Amylase. 9. Calculate the rate of substrate hydrolyzed for all four reactions in mg/sec, using the equation Rate = 10mg starch/Time in seconds. Worksheet for your α Amylase 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. Enzyme Concentration Starch added (mg) Time to Complete Reaction (sec) Reaction Rate (mg/sec) 1% 2.5% 5% 10% Notes: 9 Exercise 1 Enzymes EXPERIMENT 3: THE EFFECT OF pH ON α AMYLASE RATE 1. For this part of the experiment, you will work with the other pair of students at your lab table. Between the two groups, select one of your dilutions of α ­amylase to use for this experiment. You should use a solution that had a relatively fast reaction rate (20 sec ­2 min), but keep in mind that you will need at least 4mL of the stock for four different pH reactions, and should have more than that remaining. 2. Copy the endpoint time and rate data for the α ­Amylase pH 7.0 reaction from the α ­Amylase concentration experiment above – use this for the pH 7.0 value for this experiment. Write thes...
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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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