Exercise 2: Biologically Important Molecules
Objectives:
-Students will be able to differentiate between positive and negative controls
-Students will be able to identify/design positive and negative controls when
presented with experimental development scenarios
-Students will be able to identify and determine the structure and function of the
macromolecules and their subunits
-Students will be able to use different reagents to identify the presence of
macromolecules in known and unknown solutions.
-Students will be able to differentiate between positive and negative test for each of
the macromolecules.
-Students will be able to compare and contrast the results of each test to identify
differences in the intensity of the reactions.
-Students will be able to identify and explain whether a reaction is undergoing a
dehydration synthesis or hydrolysis reaction.
-Students will be able to differentiate between an oxidation reaction and a reduction
reaction.
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BIO-MOLECULES
READ: Introduction to Biologically Important Molecules pp.37-39
1. How do mono-, di- and polysaccharides differ in structure? Give examples of each.
2. How do mono-, di-, and polysaccharides differ in function?
Task 1- Carbohydrates
READ: pp.39-41
Perform Procedure 4.1 on pp.39- Examining Reducing Sugars using the Benedict’s
Reagent. Make sure to follow all the TABLES in the Task Sheet and NOT the lab
manual.
Record your predictions and results in Table 2.1 below
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Table 2.1 Benedict’s Test & Iodine Test
Tube
1
2
3
4
5
6
7
8
Solution
Benedict's Test Results
Expected (color)
Observed (color)
Iodine Test Results
Expected (color)
Observed (color)
10 drops onion juice
10 drops potato juice
10 drops sucrose
10 drops glucose
10 drops distilled water
10 drops reducing sugar
10 drops starch
10 drops unknown
solution
Benedict’s Test for Reducing Sugars- Questions
3. In your own words, what is a negative control?
4. In your own words, what is a positive control?
5. Why are these types of controls (positive and negative) used in experiments?
6. What observations indicate a positive test for reducing sugars?
7. Using Table 2.1, identify the positive and negative controls for the Benedict’s Test.
Explain.
8. Is there a relationship between the amount of reducing sugars in the solution and the
intensity of the reaction? Explain.
9. Will the Benedict’s Reagent detect the presence of ALL sugars? Explain.
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10. Rank the solutions from non-reducing sugar to strongest reducing sugar.
11. Based upon the results of the Benedict’s Test what can you conclude about your
unknown solution?
Perform Procedure 4.2 on pp. 41 Examining Starch using the Iodine Test
Record your predictions and results in Table 2.1 above.
Iodine Test for Starch- Questions
12. What type of carbohydrate are you testing for when you use the Iodine Test? Is this type
of carbohydrate a mono-, di-, or polysaccharide?
13. What observations indicate a positive test for starch?
14. Explain the results seen in the following solutions:
a. Onion Juice
b. Potato Juice
15. Is there a relationship between the amount of starch in the solution and the intensity of
the reaction? Explain.
16. Using Table 2.1, identify the positive and negative controls for the Iodine Test. Explain.
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17. Based upon the results of the Iodine Test what can you conclude about your unknown
solution?
Task 2- Proteins
READ: pp. 42-43
Perform Procedure 4.3 on pp.42- Examining Proteins using the Biuret’s Reagent
Record your predictions and results in Table 2.2 below
*Correction in lab manual: Procedure 4.3- Step 4: Add 2mL of 2.5% NaOH,
followed by about 1mL of Biuret’s Reagent.
Table 2.2 Biuret Test
Tube
1
2
3
4
5
6
7
Solution
2mL egg albumen
2mL honey
2mL amino acid solution
2mL distilled water
2mL 1% protein solution
2mL 3% protein solution
2mL unknown solution
Expected Results (color)
Observed Results (color)
Biuret Test for Proteins- Questions
18. What monomer comprises a polypeptide?
19. In the Biuret Test, what molecular structure produces the reaction seen in the test?
20. What observations indicate a positive test for proteins?
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21. Using Table 2.2, identify the positive and negative controls for the Biuret Test. Explain.
22. Is there a relationship between the amount of proteins in the solution and the intensity
of the reaction? Explain.
23. Compare and contrast the results seen in test tubes 3 & 5 of the Biuret Test.
24. Compare and contrast the results seen in test tubes 5 & 6 of the Biuret Test.
25. Based upon the results of the Biuret Test what can you conclude about your unknown
solution?
Lab 2 Task 3- Lipids
READ: pp. 43-44
Perform Procedure 4.4 on pp.43- Examining Lipid Solubility
Record your predictions and results in Table 2.3 below
Table 2.3 Solubility Test
Tube
1
2
3
Solution
3mL water
3mL acetone
3mL unknown solution
Expected Results
Observed Results
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Lipid Solubility- Questions
26. Do lipids dissolve in water? Why or why not?
27. Compare and contrast the results in test tube 1 & 2 of the Solubility Test.
28. What did the solubility test demonstrate about the structure and solubility properties of
a lipid?
29. Based upon the results of the Solubility Test what can you conclude about your
unknown solution?
Perform Procedure 4.5 on pp.43- Examining Lipids using the Sudan IV Reagent
Record your predictions and results in Table 2.4 below
*Correction in lab manual: Procedure 4.5- Step 4: Add 2mL of Sudan IV
Reagent to all the test tubes.
Figure 1. A stain known as Sudan IV combines with lipid molecules to produce a bright red
color. This is an example of a positive result of the Sudan IV test.
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Table 2.4 Sudan IV Test
Tube
1
2
3
4
5
Solution
1mL salad oil
1mL honey
1mL distilled water
1mL known lipid
1mL unknown solution
Expected Results (color)
Observed Results (color)
Sudan IV Test for Lipids- Questions
30. What observations indicate a positive test for lipids?
31. Using Table 2.4, identify the positive and negative controls for the Sudan IV Test.
Explain.
32. Is there a relationship between the amount of lipids in the solution and the intensity of
the reaction? Explain.
33. Based upon the results of the Sudan IV Test what can you conclude about your unknown
solution?
Task 4: Dische Diphenylamine Reagent Test- Nucleic Acids
DNA and RNA are nucleic acids made of nucleotide subunits. One major difference between
DNA and RNA is their sugar: DNA contains deoxyribose, whereas RNA contains ribose. DNA
can be identified chemically with the Dische Diphenylamine test. Acidic conditions convert
deoxyribose to a molecule that binds with diphenylamine to form a blue complex. The
intensity of the blue color is proportional to the concentration of DNA.
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Figure 2. Basic nucleic acid structure
Handle the Dische diphenylamine reagent carefully; it is toxic. Use gloves
when handling the reagent and keep the stock solution under the hood.
Procedure: The Dische diphenylamine test for DNA
1. Half fill a 250mL beaker with water. Place it on a hot plate at your station and allow it to
come to a gentle boil.
2. Obtain four test tubes and number them 1-4.
3. Add the materials listed in Table 2.5.
4. Add 2 mL of the Dische diphenylamine reagent to each tube and mix thoroughly.
5. Place the tubes in a boiling water-bath for 10 minutes.
6. In the meantime, predict the color changes you expect to occur in each test tube and
record them in Table 2.5. Also mark which tube you think is the positive control with an
* and which is the negative control with a **.
7. After 10 minutes, transfer the tubes to an ice bath. Gently mix and observe the color of
their contents as the tubes cool. Record your observations in Table 2.5.
*Dispose of all solutions in the Dische waste container in the hood.
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Table 2.5 Dische Diphenylamine Test
Tube
1
2
3
4
Solution
2mL DNA solution
1mL DNA solution +
1mL water
2mL Distilled water
2mL Unknown Solution
Expected Results (color)
Observed Results (color)
Dische Diphenylamine Test for DNA- Questions
34. What observations indicate a positive test for DNA?
35. Using Table 2.5, identify the positive and negative controls for the Dische
diphenylamine Test. Explain.
36. Is there a relationship between the amount of DNA in the solution and the intensity of
the reaction? Explain.
37. Would the Dische diphenylamine Reagent be a good indicator for the presence of RNA?
Explain.
38. Based upon the results of the Dische Diphenylamine Test what can you conclude about
your unknown solution?
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