Group C -Amylase Lab


Part C. Effect of different amounts of substrate (amylopectin)

1. Select 5 test tubes labeled C1 through C5 and place them in a test tube rack. Be sure to label test tubes with a permanent marker.

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.

Test tube #
DI Water
Amylopectin
Amylase
C1
1000 µl

.

0 µl

.

0.2 ml

.

C2
900 µl

.

100 µl

.

0.2 ml

.

C3
800 µl

.

200 µl

.

0.2 ml

.

C4
400 µl

.

600 µl

.

0.2 ml

.

C5
0 µl

.

1000 µl

.

0.2 ml

.

3. Using the adjustable pipetter, add the amount of amylopectin solution (substrate) indicated in the table to each tube. CAUTION: Be careful to fully add the small volumes of C2.

4. Using a pipette pump dispenser, add 0.2 ml of amylase solution to each tube as quickly as possible. Note the time when the amylase is added to the last tube.

5. After exactly 10 minutes, use the pipette 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 at once.

6. Mix the contents of each tube using the laboratory mixer and place each tube into a hot (use caution) water bath for five minutes. Check off each step in the table below.

Test Tube #
DNS Solution
Tartrate Solution
Vortex Mixer ()
Hot Water Bath
()
Absorbance Measured
Conversion to µl of Maltose
C1
1 ml

.

1 ml

.

.

.

.

.

C2
1 ml

.

1 ml

.

.

.

.

.

C3
1 ml

.

1 ml

.

.

.

.

.

C4
1 ml

.

1 ml

.

.

.

.

.

C5
1 ml

.

1 ml

.

.

.

.

.

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 your 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 amylopectin solution added as indicated in the worksheet.


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