Group B -Amylase Lab

Part B. Effect of different amounts of amylase

1. Select 5 test tubes, label B1 through B5 and place them in a test tube rack.

2. Using an adjustable pipetter, add the amount of deionized water indicated in the table below to each tube. Check off each chemical as you add it.

3. Using an adjustable pipetter, add the amount of amylase indicated in the table to each tube. CAUTION: Be careful to fully add the small volumes used for test tube B2.

Test tube #
DI Water
Amylase
Amylopectin
B1
1000 µl

.
0 µl

.
1 ml

.
B2
950 µl

.
50 µl

.
1 ml

.
B3
900 µl

.
100 µl

.
1 ml

.
B4
700 µl

.
300 µl

.
1 ml

.
B5
0 µl

.
1000 µl

.
1 ml

.

CAUTION: The next step is important. Please read and follow the directions carefully.

4. Using a pipette pump dispenser, add 1 ml of amylopectin to each tube. Add the amylopectin solution as quickly as possible to all tubes and note the time when the solution is added to your last tube.

5. After exactly 10 minutes, use the pipette pump dispensers to add 1 ml of DNS reagent and 1 ml tartrate solution to each tube. Caution: DNS reagent should be washed off your skin immediately.

Test Tube #
DNS Solution
Tartrate Solution
Vortex Mixer ()
Hot Water Bath
()
Absorbance Measured
Conversion to µl of Maltose
B1
1 ml

.
1 ml

.

.

.

.

.
B2
1 ml

.
1 ml

.

.

.

.

.
B3
1 ml

.
1 ml

.

.

.

.

.
B4
1 ml

.
1 ml

.

.

.

.

.
B5
1 ml

.
1 ml

.

.

.

.

.

6. Mix the contents of each tube using the vortex mixer and place them into a hot (use caution) water bath for five min. Check off each step as you complete it in the table above.

7. Remove the tubes from the water bath and allow them to cool to room temperature. Observe and make note of the color change - use color pencils if available. Store the tubes in the refrigerator until the next lab period.

Next Day or if time permits:

8. Take your samples to the spectrophotometer. Measure the absorbance at a wavelength of 540 nm of the contents of each tube. Record the absorbance in the above table. Your teacher will provide instructions on the use of the spectrophotometer. The absorbance is proportional to the amount of maltose produced by the enzyme in ten minutes.

9. Prepare a graph of absorbance versus volume of amylase solution added as indicated in the worksheet.

Go to directions for Part [A] [C] [D]