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• Ap Biology Lab Manual Lab 111 2
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Introduction:
Cellular respiration is the release of energy from organic compounds by metabolic chemical oxidation in the mitochondria within a cell. There are a number of physical laws that relate to gases and are important in the understanding of how the equipment in this lab works. These are summarized as general gas laws that state: PV=nRT where: P stands for pressure of the gas, V stands for the volume of the gas, n stands for the number of molecules of gas there are, R stands for the gas constant, and T stands for the temperature of the gas. A respirometer is the system used to measure cellular respiration. Pressure changes in the respirometer are directly relative to a change in the amount of gas there is in the respirometer as long as the volume and the temperature of the respirometer do not change. To judge the consumption of oxygen in two different respirometers you must reach equilibrium in both respirometers.

Cellular respiration is the procedure of changing the chemical energy of organic molecules into a type that can be used by organisms. Glucose may be oxidized completely if an adequate amount of oxygen is present. The equation for cellular respiration is C6H12O6 + 6O2 à 6CO2 + 6H2O + energy. Carbon dioxide is formed as oxygen is used. The pressure due to CO2 might cancel out any changes due to the consumption of oxygen. To get rid of this problem, a chemical will be added that will selectively take out the carbon dioxide put off. Potassium hydroxide will chemically react with the carbon dioxide by this equation: CO2 + K2CO3 + H2O.

Hypothesis:
The rate of cellular respiration will be higher in germinating peas in cold and room temperature water baths than in that of the beads or non-germinating peas. The cooler temperature in the cold water baths should slow the process of cellular respiration in the peas.

Oct 19, 2017 AP Biology Syllabus, Class Procedures Course Description. AP Biology Tentative Lesson Plans; AP Bio Summer Assignment 2019-2010; AP BIOLOGY Chapter powerpoints; AP bio Reading guides Biology in Focus 2nd edition; AP Biology Labs; How to write a lab proposal, formal lab, and primers on statistics; AP Biology 'Doing Biology' AP bio cell.

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Materials:
The materials used in this lab were the following: a water bath, a graduated cylinder, a thermometer, tape, metal washers, beads, germinating peas, non-germinating peas, beads, beakers, another graduated cylinder, ice, paper, and a pencil are needed for this lab.

Methods:
Obtain a room temperature water bath and a 10-degree Celsius water bath. Add ice to room temperature water and watch the thermometer until the temperature has reached 10-degrees Celsius. For respirometer one, obtain a graduated cylinder and fill it with 50mL of water. Drop in 25 germinating peas and determine the water displacement. Record the volume, remove the peas and place them on a paper towel. For respirometer two, obtain the same graduated cylinder, filled again with 50mL of water. Drop twenty-five of the non-germinating peas in the water and continue adding beads to the water until the same water displacement for the non-germinating peas equals the first result. Remove the contents, and drain the water leaving the peas and beads to dry on a paper towel. For respirometer three, fill the 100mL graduated cylinder with 50mL of water and obtain the first water displacement value by adding just beads to the water in the cylinder. Take out the beads, allow the water to drain, and repeat this same procedure for respirometers 4, 5, and 6, which will be placed in the cooler water. For assembly of the respirometers, obtain 6 vials, each with a stopper and a pipette. Place a small wad of absorbent cotton in the bottom of each vial and using a dropper, saturate the cotton with 15% KOH solution. Make sure the vials are dry on the inside. Do not get KOH on the sides of the respirometer. Place a small wad of non-absorbent cotton on top of the KOH saturated cotton, making sure the same amount is used for each respirometer. Place the first set of peas in their respective vials. Do the same for the second set of peas. Insert the stopper with the calibrated pipette. Place a weighted collar on the end of each vial. Make a sling of masking tape attached to each side of the water baths to hold the pipettes out of the water during the equilibration period of seven minutes. Vials 1, 2, and 3, should rest in the room temperature water while 4, 5, and 6, should rest in the 10-degree Celsius water bath. After seven minutes of equilibration, immerse all 6 respirometers entirely in their designated water baths. Water enters the pipette for a short distance and stops. If the water continues to move into a pipette, check for leaks. Working quickly, arrange the pipettes so the can be read through the water at the beginning of the experiment. These should not be shifted during the experiment. Keep hands out of the water bath after the experiment has started. Make sure a constant temperature is maintained. Allow respirometers to equilibrate three more minutes, record the initial position of the water in each pipette to the nearest .01mL. Check the temperature in both water baths and record in table 5.1. Check and record every five minutes for twenty minutes by repeating the procedure for that task.

Data:

Table 5.1

Graph 5.1

Table 5.2

Graph 5.2

Questions:
In this activity, you are investigating both the effects of germination versus non-germination and warm temperature versus cold temperature on respiration rate. Identify the hypothesis being tested on this activity. The hypothesis of this experiment was: The rate of cellular respiration will be higher in germinating peas in cold and room temperature water baths than in that of the beads or non-germinating peas. The cooler temperature in the cold water baths should slow the process of cellular respiration in the peas.

This activity uses a number of controls. Identify at least three of the controls, and describe the purpose of each. First of all, the water baths held a constant temperature. Secondly, the volume of KOH was constant from vial to vial. Lastly, the equilibration period was identical for all the respirometers.

Describe and explain the relationship between the amount of oxygen consumed and time. The amount of oxygen that was consumed was the greatest in the warmer water. The oxygen consumption increased over time in the germinating peas.

Why is it necessary to correct the readings from the peas with the readings from the beads? This is necessary to show the actual rate at which cellular respiration occurs in peas. The beads served as a control variable.

Explain the effects of germination versus non-germination on peas seed respiration. The germinating seeds are alive and growing, therefore respirate to grow.

Explain the results shown in the sample graph in your lab manual. As the temperature increased, the enzymes denatured so germination was inhibited.

What is the purpose of KOH in this experiment? The KOH drops absorbed the carbon dioxide so that it would not cause the put off of that gas to make the readings equilibrate.

Why did the vial have to be completely sealed under the stopper? The stopper at the top of the vial had to be completely sealed so that no gas could leak out of the vial and no water would be allowed into the vial.

If you used the same experimental design to compare the rates of respiration of a 35g mammal at 100 degrees Celsius, what results would you expect? Explain your reasoning. Respiration would be higher in the mammal since they are warm-blooded.

If respiration in a small mammal were studied at both room temperature, 21-degrees Celsius and 10-degrees Celsius, what results would you predict? Explain your reasoning. Respiration would be higher at 21 degrees because the animal would have to keep its body temperature up. The results would multiply at 10-degrees because the mammal would have to keep its body that much warmer in comparison to the room temperature.

Explain why water moved into the respirometer pipettes. While the peas underwent cellular respiration, they consumed oxygen and released carbon dioxide which reacted with the KOH in the vial, resulting in a decrease of gas in the pipette. The water moved into the pipette because the vial and pipette were completely submerged into the bath.

Design an experiment to examine the rates of cellular respiration in peas that have been germinating for 0, 24, 48, and 72, hours. What results would you expect? Why? A person could set up four respirometers which have one of the following: Seeds that have not begun to germinate, seeds germinating for one day, seeds germinating for two days and seeds germinating for three days. The results would probably be that there would be no oxygen used by the seeds that have not germinated yet. The seeds that have been germinating for three days will have the greatest amount of oxygen consumption.

Error Analysis:
Error in this lab could have occurred if the seals on the vials weren’t tight and there was a leak of water into the vials. Another source of error could have been if the KOH had touched the sides of the vial. Also, the absorbent cotton balls that were used for the KOH could have been too saturated. Another source of error could be at the temperature of the water baths. If a close eye wasn’t kept on the temperature, the ten degree Celsius would have fallen in degrees.

Conclusion:
Oxygen consumption in respirometers with germinating peas is greater than in the respirometers with non-germinating peas. Respiration was affected by the temperature of the water bath as well. Respiration occurs faster in the warmer water baths.

Completing the Research Notebook for AP Biology Lab #6.....Molecular Biology

Resource: Lab Six, Molecular Biology

Pages 64-77 in the AP Biology Lab Manual

Note: you will be using handouts from Carolina Biological kits for this laboratory procedure.

Ap Biology Lab Manual Lab 111 2

Part 1: Title

Develop a title in the form of a question after completing the pre-lab.

Part 2: Objectives (What are the objectives for this laboratory?)

Part 3: Prelab Questions

Transformation

1. Describe E. coli and its singular circular chromosome.

2. Explain plasmids, including “R” plasmids.

3. Explain 3 ways that genes can be transferred between bacteria.

4. Explain what is meant by “competent”.

5. Explain figure 6.1

6. Figure 8 is the plasmid we will be using to transform. Inside the circular plasmid there are two bold, thick black

semicircles. These are the two genes on this plasmid that we are interested in. What are they?

7. What is “X gal” and what will happen if bacteria are transformed with this plasmid?

Gel Electrophoresis

8. What are restriction endonucleases (enzymes)?

9. Explain how they are named (EcoRI)???

10. Explain Figure 6.2b (page 69, AP Biology Lab Manual)

11. In this experiment, how will the three samples of DNA be cut?

12. Explain what causes the DNA to “move” through the agar gel in the gel chamber and what causes the

separation of the bands into “DNA fingerprints”.

IT IS VERY IMPORTANT THAT YOU READ THESE PAGES AT LEAST TWICE.

THE PROCEDURE IS VERY COMPLICATED, AND YOU NEED TO BE PREPARED.

TRANSFORMATION: READ PAGES 4 AND 5 (procedure from Carolina Biological kit)

ELECTROPHORESIS: READ PAGES 10, 11, 12 (procedure from Carolina Biological kit)

Part 4: Method

Transformation: Starter Plates of E. coli will be obtained which have a source of bacterial cells in an exponential

growth phase. The plasmid used is pBLU which has a gene for ampicillin resistance and a gene which will cause a

chemical called X gal to form blue colonies. In a controlled experiment, the E. coli bacteria cells will be made

“competent” by suspending them in cold calcium chloride, incubating them on ice, adding the plasmid, heat shocking

them, and then subjecting them to ice. The cells will be spread on luria broth agar with and without ampicillin for 24

hours in an incubator.

Gel Electrophoresis: A gel casting tray will be sealed on each end with masking tape, liquid agar will be poured into

the tray and allowed to solidify. The tray will be placed in a gel box so that the comb is on the “negative” or black

end, and TBE buffer will be poured over the agar. The comb will be removed. The box will be covered with wrap.

The next morning, micropipets will be used to transfer DNA subjected to various restriction enzymes into the wells,

and the power source will be turned on.

Part 5: Data

Design data tables that will enable you to collect qualitative and quantitative data from both procedures:

transformation and gel electrophoresis of DNA.

Post-lab:

Part 6: Questions

The questions will be found on student guides from the Carolina Biological kits for these labs.

Page 6 and 7 #1-10

Page 12 and 13 # 1-4

Part 7: Graph

You will need to construct a graph in order to determine the number of base pairs for the fragments with the EcoRI

restriction enzyme cut in the DNA gel electrophoresis lab.

The graph paper used is semilog graph paper. Directions will be reviewed during class. The directions are on the

student guide for the lab. The directions are also on page 72-73 of the AP Biology Lab Manual.

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Part 8: Theme Correlation

Explain how this lab demonstrated both inquiry and structure fits function.

Inquiry: How was the transformation experiment controlled? Was biotechnology demonstrated?

Structure Fits Function: How can genes of interest be placed into plasmids?

Part 9: Conclusion

1. Use the transformation lab results to explain how plasmids can be used in genetic engineering.

2, Gel electrophoresis: Use the lab results to explain how restriction enzymes work; the function of an electrical

field during gel electrophoresis; the function of the agarose gel.

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3. What is a restriction site? How could a mutation that alters a restriction site be detected by gel electrophoresis?