AP Biology

2021-22
Lab Manual

Hour: .
Name: .
 Table of Contents
Lab write-up Format 2
Cellular Respiration Activity 5
Pre-Lab Questions: 6
Data Collection: 6
Data Analysis: 7
Conclusion: 8
Enzyme Pre-Lab 10
Procedure: 11
Questions: 12
Enzyme Lab Procedure 13
Data Collection: 14
Post-Lab Questions: 15
Cell Size Lab 17
Results: 18
Questions: 18
Diffusion & Osmosis Lab 19
Dialysis Tubing Procedure 20
Data Collection & Analysis 22
Conclusion Questions 22
Observing Osmosis in Living Cells 24
Design an Experiment 25
DNA Replication On the Floor 26
Setup 26
Replication 27
Gel Electrophoresis Lab 30
Questions: 31
Pill Bug Behavior Choices 33
Data Tables 34
Analysis and Discussion 34
pGLO Transformation Lab 36
Lesson 1 Introduction to Transformation 36
Lesson 1 Focus Questions 37
Lesson 2 Transformation Laboratory 40
Transformation Procedure: 41
Lesson 2 Review Questions 45
Lesson 3 Data Collection and Analysis 46
A. Data Collection 46
B. Analysis of Results 47
Lesson 3 Review Questions 48

‘21-’22 AP Biology Lab Manual 1

 Lab write-up Format
Title – Center at the top of paper.

*Skip a Line *

Purpose: Clearly state in one or two sentences what you are trying to determine in the lab
experiment.

Procedure: Using complete sentences clearly state how the experiment was done. If the
procedure is very long and complex, summarize it.
Should be organized as bullets or steps not in paragraph or story form.

***Include a labeled diagram or picture of any lab set up.****

Data: Data can be qualitative or quantitative. Regardless of the type of data, it should always be
organized in a data table format for quick reference.
***Your numbers should always be in columns.***
Example:

Good example Bad example

mass of Volume of
metal (g) metal (ml) grams milliters

Trial 1 Trial 1

Trial 2 Trial 2

Calculations: All numbers in a calculation should be clearly labeled and units recorded.
Example:

Mass of salt solution: (use headings for each calculation and skip a line between calculations)
49.38g - 32.05g = 17.33g

Questions: Not all labs have a questions section but if the lab does contain a questions section
make sure to include it along with the proper formatting.

Answers to questions should be in complete sentences and the question should be restated in
your response. Don’t recopy the Questions!!

Example: question – How does your calculated answer compare with the accepted
value?
Response – My calculated value is slightly higher than the accepted value. The
difference in the value is most likely caused by the lack of precision in the graduated
cylinder we used. The lack of precision and rounding caused our experimental values to
be higher than what was expected.

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Conclusion: Your conclusion is the place you will summarize the entire lab. As such you
should start your conclusion by establishing why the lab was done (purpose) or what you hoped
to learn from it. Follow that up with any important background information or what you thought
might happen based on prior knowledge (hypothesis / background).

The next section showcases your data. You should include your actual results along with what
they suggest or support. Connect the dots for your reader. Why does this data support your
purpose or why might it contradict what you thought might happen. Did it confirm what you
thought or what we learned in class or didn’t your lab results fit with what you were thinking?

Your conclusion should end with a discussion about possible sources of error in the lab.
Specifically systematic error, could the lab have been set up differently to get better results?
Was there a part of the lab that was specifically difficult to get accurate results and is there a
way to gather the same information in a more efficient and reliable manner? Some labs go well
with no apparent errors, which can be stated. You can also end your lab with what the next step
might be. If your results uncover more questions than answers, what line of experiments might
shed some light on the new problems discovered?

•In lab reports I am looking for your best work. They take some time to complete and you should
take pride in doing them as they are a reflection of the work that you can produce.

•Use the format above to help you gain all the appropriate points for the format.

‘21-’22 AP Biology Lab Manual 3

 Cellular Respiration Activity
The purpose of this lab is to get you familiar with being back in a lab setting and work on your
problem solving skills right away in the school year. The materials that you need and the
procedure you will follow are written below. It is the responsibility of you and your lab partners to
complete this lab activity and

Problem:
How does exercise affect disposal of waste (CO2) from cellular respiration?

Materials:
● 6 flasks
● 50 ml beaker
● 5 ml bromothymol blue per student
● 10 ml, 100 ml graduated cylinders
● 6 straws
● clock or stopwatch

Procedure:

Directions: Circle any measurements you need to make.

1. You will complete this lab in a group of four: two people are the recorders and the other
two will perform the exercise.
2. Record your hypothesis in the pre-lab questions below, as to how exercise will affect
your body’s production of CO2
3. Label 6 flasks A-F. Put 50 ml of water and 5 ml of bromothymol blue in each flask.
4. Bromothymol blue (BTB) is an indicator of pH. When CO2 is bubbled through
later, carbonic acid is formed. CO2 + H2O ⟶H2CO3. BTB changes color toward
yellow in the presence of an acid. The more yellow the BTB is, the more carbon
dioxide is present.
5. When the recorders say ‘go’, the performers should gently blow air through the straw
into the bottom of flasks A (person 1) and B (person 2).
CAUTION: Do not inhale through the straw!
6. The recorders will record the time it takes for the color to change from BLUE to
YELLOW.
7. Now the performers should jog in place for 1 minute and repeat steps 3 and 4 using
flasks C and D.
CAUTION: If you feel dizzy or weak while exercising, stop immediately and sit
down.
8. Finally – the performers should sprint or run for 1 minute and repeat steps 3 and 4 using
flasks E and F.

‘21-’22 AP Biology Lab Manual 4

Pre-Lab Questions:
1. What is the independent variable: ___________________________ (x-axis)
2. What is the dependent variable: ____________________________ (y-axis)
3. What is the control group?

a. Why do you need this control group:

4. What are three constants in this experiment?

5. What SAFETY precautions do you need to take in this lab?

6. What is your hypothesis? (be sure to follow the “If....Then….Because….” format from
Biology 1)

Data Collection:

Person #1 Time for solution to Person #2 Time for solution to

Results change color(sec) Results change color(sec)
Before Before
exercise exercise
After Light After Light
exercise exercise
After Intense After Intense
exercise exercise

1. Your group’s average time before exercise:

2. Your group’s average time after light exercise:

3. Your group’s average time after intense exercise:

‘21-’22 AP Biology Lab Manual 5

 4. Average time before exercise of three other groups:

1) 2) 3)

5. Average time after light exercise of three other groups:

1) 2) 3)

6. Average time after intense exercise of three other groups:

1) 2) 3)

Data Analysis:

Plot the average time before and after exercise of your group as compared to three other
groups’ average times. This is a BAR GRAPH, and you should plot 12 different bars (1 Ave
before exercise, and 2 Averages after exercise). Remember to label your X-, Y-axis, and
include a title for your graph.

‘21-’22 AP Biology Lab Manual 6

What process in your body produces carbon dioxide?

How does exercise affect this process? How do you know? Cite your data.

Was there a difference between the two experimental groups? Explain why or why not, citing
your data.

Conclusion:

Address your hypothesis:

How did exercise affect the time for the BTB solution to change color?

Did these results support/reject your hypothesis?

How were you able to determine the amount of CO2 that was produced? Describe the process.

Why is cellular respiration important to your body?

‘21-’22 AP Biology Lab Manual 7

What would happen if your cells were not getting enough oxygen during the running portion of
the lab? Explain in 2-3 full sentences. What are the products of this reaction?

Error analysis: What do you think are potential sources of error? List at least 3.

‘21-’22 AP Biology Lab Manual 8

 Enzyme Pre-Lab
2.3: Describe the role of energy in organisms.

Directions:
Read all background information, materials, and procedure. It is important to understand
generally how to perform the experiment before starting this experiment, since it is time
sensitive! You will also be provided with the procedure when you perform the lab, so you will not
need this Pre-Lab when it comes time to perform the experiment.

Background Information:
You have been learning about enzymes in class. Remember that enzymes are proteins that
speed up reactions by lowering the activation energy required to begin a chemical reaction. In
this lab we will be using the enzyme, Peroxidase, which are found in animals, plants, and
bacteria. Their job is to protect cells against the effects of oxidative stress (don’t worry about
what “oxidative” means- we will talk about it next unit) and cell damage due to hydrogen
peroxide. Hydrogen peroxide is a byproduct of cellular respiration. Since it is toxic to cells, it
needs to be broken down, which is where Peroxidase comes in. Peroxidases are easily
extracted from turnips, which is where we will be getting our Peroxidases
from. We can use them to study the rate of reaction depending on different
factors, such as environmental factors that you have learned in lecture (pH,
temperature, enzyme and substrate concentration). Peroxidase is used to
catalyze the reaction of hydrogen peroxide into water so that it cannot harm
cells.

You will be measuring the absorbance of the orange product formed from
guaiacol and hydrogen peroxide (as the reaction goes on, the solution
becomes more and more orange) as a function of time for three different
concentrations of turnip peroxidase.

Spectrophotometers (pictured below) is an instrument used to measure the amount of light

that is absorbed by a material. Material (in this case, we will put peroxidase and hydrogen
peroxide (along with other solutions) into a cuvette (pictured right)
which is then placed inside the spectrophotometer, and it
measures how much light is absorbed.

*Below are two images of the spectrophotometers we have. We

have an older model (left) and a newer model (right).

‘21-’22 AP Biology Lab Manual 9

Materials Your Lab Group Will Need:
1. 16 mL of Buffer, pH 5
2. 4 mL Guaiacol 0.2% solution in isopropyl alcohol
3. 8 mL Hydrogen peroxide 0.02% solution
4. 8 mL phosphate buffer, pH 7
5. 6 mL peroxidase enzyme extract in phosphate buffer
6. 5, 2 mL pipettes
7. Spectrophotometer
8. 7 Test tubes (13 x 100 mm)
9. Timer (one person in your group can grab your phone)

Procedure:
1. Make sure you thoroughly read and understand the ENTIRE procedure before
beginning, since timing is crucial for this experiment to work.
2. Ms. Ingalls has already turned the spectrophotometer on, and set it to 500 nm. It takes
spectrophotometers about 20 minutes to warm up.
3. Prepare a blank cuvette by combining 3 mL pH 5 buffer, 2 mL 0.02% hydrogen peroxide,
1 mL 0.2% guaiacol, and 2 mL phosphate buffer in a test tube.
4. Zero the spectrophotometer using the blank solution.
5. Prepare three test tubes containing substrates (one tube will be labeled “S1”, the next
tube will be labeled “S2” and the last tube will be labeled “S3”)
6. Prepare three test tubes containing enzymes (one tube will be labeled “E1”, the next
tube will be labeled “E2”, and the last tube will be labeled “E3”
7. In S1, S2, and S3, add:
a. 1 mL pH 5 buffer
b. 2 mL 0.02% Hydrogen peroxide
c. 1 mL 0.2% Guaiacol
8. In E1, add:
a. 2 mL pH 5 buffer
b. 0.5 mL enzyme extract
c. 1.5 mL phosphate buffer
9. In E2, add:
a. 2 mL pH 5 buffer
b. 1 mL enzyme extract
c. 1 mL phosphate buffer
10. In E3, add:
a. 2 mL pH 5 buffer
b. 0.25 mL enzyme extract
c. 1.75 mL phosphate buffer
11. When ready to begin trial 1, pour the contents of S1 tube into E1 tube, and
IMMEDIATELY start timing. Pour the combined contents into a cuvette, place in the
spectrophotometer (so yes, you will begin timing before you even put the cuvette in the
spectrophotometer.
12. Measure and record the absorbance every 20 seconds for a total of 5 minutes.

‘21-’22 AP Biology Lab Manual 10

 13. Repeat steps 11 and 12 for the next 2 series of tubes.
14. Graph the absorbance versus time for each trial on one graph to determine the rate of
reaction.

***We only have two spectrophotometers, so groups will have to go at separate times. But all
lab groups can set up their test tubes at the same time.

Questions:
**These should be done before the lab**
1. What is the scientific question that we are trying to answer?

2. What is your hypothesis for this experiment?

3. How do enzymes work?

4. What is the purpose of placing the enzymes and hydrogen peroxide with guaiacol in the
spectrophotometer? (What will it tell us?)

‘21-’22 AP Biology Lab Manual 11

 Enzyme Lab Procedure
Safety
1. Guaiacol is toxic by ingestion. It is aromatic and can be irritating to the nose and throat.
2. Isopropyl alcohol is flammable.
3. Hydrogen peroxide can be irritating to eyes and skin.
4. Wear goggles.
5. Wear gloves.
6. Wash hands when done.

Procedure:
1. Make sure you thoroughly read and understand the ENTIRE procedure before
beginning, since timing is crucial for this experiment to work.
2. Ms. Schwobe has already turned on the spectrophotometer, and set it to 500 nm. It
takes spectrophotometers about 20 minutes to warm up.
3. Prepare a blank cuvette by combining 3 mL pH 5 buffer, 2 mL 0.02% hydrogen peroxide,
1 mL 0.2% guaiacol, and 2 mL phosphate buffer in a test tube.
4. Zero the spectrophotometer using the blank solution. (or put the blank in and click “cal”
for the colorimeter)
5. Prepare three test tubes containing substrates (one tube will be labeled “S1”, the next
tube will be labeled “S2” and the last tube will be labeled “S3”)
6. Prepare three test tubes containing enzymes (one tube will be labeled “E1”, the next
tube will be labeled “E2”, and the last tube will be labeled “E3”
7. Set the test tubes in the rack in their pairs. S1 with E1, S2 with E2, S3 with E3.
8. Obtain a graduated cylinder with 7 mL pH 5 buffer. Go back to your station and measure
out the correct amount in each test tube. The “recipes” are below.
9. Rinse your graduated cylinder, then fill it with 6 mL Diluted Hydrogen Peroxide. Go back
to your station and measure out the correct amount in each test tube. The “recipes” are
below.
10. Rinse your graduated cylinder, then fill it with 3 mL Guaiacol. Go back to your station
and measure out the correct amount in each test tube. The “recipes” are below.
11. Rinse your graduated cylinder, then fill it with 1.75 mL of enzyme extract. Go back to
your lab station and measure out the correct amount in each test tube. The “recipes” are
below.
12. Rinse your graduated cylinder, then fill it with 4.25 mL of phosphate buffer. Go back to
your lab station and measure out the correct amount in each test tube. The “recipes” are
below.

In S1, S2, and S3, add: In E1, add: In E2, add: In E3, add:
1 mL pH 5 buffer 2 mL pH 5 buffer 2 mL pH 5 buffer 2 mL pH 5 buffer
2 mL 0.02% Hydrogen peroxide 0.5 mL enzyme extract 1 mL enzyme extract 0.25 mL enzyme extract
1 mL 0.2% Guaiacol 1.5 mL phosphate buffer 1 mL phosphate buffer 1.75 mL phosphate buffer

‘21-’22 AP Biology Lab Manual 12

 13. When ready to begin trial 1, pour the contents of S1 tube into E1 tube, and
IMMEDIATELY start timing. Pour the combined contents into a cuvette, place in the
spectrophotometer (so yes, you will begin timing before you even put the cuvette in the
spectrophotometer.
14. Measure and record the absorbance every 20 seconds until your table is filled.
15. Repeat steps 18 and 19 for the next 2 series of tubes.
16. Graph the absorbance versus time for each trial on one graph to determine the rate of
reaction.

Data Collection:

Record the absorbance every 20 seconds for 4 minutes for each trial.

Seconds S1 & E1 S2 & E2 S3 & E3

20

40

60

80

100

120

140

160

180

200

220

240

‘21-’22 AP Biology Lab Manual 13

Post-Lab Questions:
1. Create a graph showing the change in absorbance for all three trials. Use the average
times for each trial and you can use Google Sheets to make the graph.

2. At what point did the rate seem to “level off”?

3. Referring to the question above, what do you think is the cause of that?

***It’s important to know that “leveling off”

is termed “Vmax”, this is when the enzymes
have reached their maximum rate of
reaction. You should be able to use this
term when answering questions and
analyzing graphs about enzyme reactions.

4. What is the Vmax of the graph to the

right?

‘21-’22 AP Biology Lab Manual 14

5. If you were to do your own experiment, what variable would you want to test?

a. Write a hypothesis for an experiment testing that variable.

b. On the graph above, predict what the rate of reaction would look like without the
presence of enzymes. Draw a line to show your prediction.

‘21-’22 AP Biology Lab Manual 15

 Cell Size Lab

Background Information:
● Iodine is an indicator for starch and turns dark purple/black.
● Cells require nutrients in order to survive.
● Cells must eliminate waste in order to survive.

Purpose:
To provide evidence for why cells are so small.

Hypothesis:
Make a statement as to which potato (cell) will have the nutrients diffuse the farthest
proportionally (closest to the center of the cell) in 20 minutes, and why do you think so.

Materials:
● Ruler
● Calculator
● Scalpel
● Potato pieces
● Paper towels
● 50mL beaker
● Water
● Iodine solution

Math Formulas:
* Surface area (mm2) = Length X Width X 6 (# of sides)
* Volume (mm3) = Length X Width X Height
* Surface area to volume ratio (mm2)/(mm3) = Surface area (mm2)

Procedure:
1. Get 3 potato cubes from the side counter, one of each size (.5cm, 1cm and 2cm)
2. Add 30mL of water to the 50 mL beaker. Have Ms. Schwobe add 1mL of iodine to the
beaker for you. Submerge the potato cubes (cells) into the iodine solution for 20
minutes.
3. While you are waiting for 20 minutes, calculate the surface area, volume, and surface
area to volume ratio for each of the 3 potato cubes (cells).
4. Carefully remove the potato cubes (cells) from the iodine solution and place them on
absorbent paper towels.
5. Cut each cell in half and observe the inside.
6. Draw each cell in detail showing the pattern of iodine throughout the potato.

‘21-’22 AP Biology Lab Manual 16

Results:

Cell Dimensions Surface area Volume (cm3) Surface Area/Volume

(cm2) Ratio

.5cm X .5cm X .5cm

1cm X 1cm X 1cm

2cm X 2cm X 2cm

Questions:
1. What do the cubes represent?

2. What does the iodine represent?

3. Why did the diffusion of iodine into the potato cube cause the color change from white to

purple?

4. What can you say about the surface area to volume ratio that will best meet the needs of

living cells?

‘21-’22 AP Biology Lab Manual 17

 Diffusion & Osmosis Lab
Read the AP lab posted in Google Classroom and use what you read to fill in the following
information.

Pre-Lab Questions – Note- the questions are not in order because I want you to actually
read the info, not just skim for answers.
1. Water can move through cell membranes by the process of
________________________. They can also move by diffusion through a certain
protein called an _____________________.

2. What does it mean when a plant cell “plasmolyzes?”

3. Draw the three beakers below, each with a cell (the circle inside) and the words
underneath each beaker. Draw each situation as prompted. Use triangles to represent
solute molecules.

4. What one factor affects osmosis in animal cells? __________________

5. Which two factors affect osmosis in plant cells? ______________________ and

_______________________

6. Describe the water potential of an isotonic solution.

7. Why does the cell wall of a plant cause turgor pressure?

‘21-’22 AP Biology Lab Manual 18

 8. What is an aqueous solution?

9. Why is the water potential of pure water in an open beaker 0?

10. What are the two components of water potential?

11. Calculate the solute potential of a 0.1M NaCl solution at 25oC.

12. If the concentration of NaCl inside an animal cell is 0.15M, which way will the water
diffuse if the cell is placed in a 0.1M NaCl solution in an open beaker?

Dialysis Tubing Procedure

Purpose:
To use dialysis tubing and various solutions to model osmosis and
diffusion.

Materials:
Read the procedure on the next page to figure out what they are, as
I’ve changed the steps a bit--list the materials below.

‘21-’22 AP Biology Lab Manual 19

Hypothesis:
You will make a hypothesis for each solution later. You do not need to put anything here for
now.

Procedure:
Note that this varies slightly from the manual. Read the steps and illustrate what you’re doing in
the space below.
1. Obtain 6 pieces of dialysis tubing (each should be about 20cm long).
2. Soak the dialysis tubing in water for about a minute. This makes them easier to open.
3. Tie off one end of the tubing, like a balloon, to make a bag. Open the other end of the
“bag.”
4. Fill the tubing with about 10mL distilled water. Then, like a balloon, tightly tie off the
other end. Cut off any extra tubing (get as close to the knot as possible).
5. Dry off the tubing and get the initial weight of each. Record. Repeat for all six tubes.
6. Obtain 6 beakers and label each with the solutions identified in the chart.
7. Fill each beaker with its respective solution- about 50 mL. Don’t put the tubing in yet!!!
8. Predict if water will enter (+) or leave (-) each bag.
9. When ready, place all six tubes, one in each beaker, at the same time. Start your timer.
10. Wait ten minutes.
11. After ten minutes, take all tubes out at the same time. Gently rinse and dry each tube.
12. Find the final mass of each tube and record.
13. The tubes can be thrown in the garbage. Rinse the beakers and clean up your station.
14. Calculate the % change for each tube. Add your % change for each solution to the data
table on the board.
15. Answer the conclusion questions.

Procedure Illustration:

‘21-’22 AP Biology Lab Manual 20

 Data Collection & Analysis

Table 1: Data Collection for the various tube/beaker pairings.

Setup Tube Beaker Predict Initial Final % Change Class average for
# Sol’n Sol’n gain/loss Weight of Weight of (final-initial)/initial * % change
of weight tube (g) tube (g) 100
(+/-)
1 10 mL 50mL
distilled distilled
water water

2 10 mL 50mL
distilled 1M NaCl
water solution
3 10 mL 50mL
distilled 1M
water Glucose
solution
4 10 mL 50mL
distilled 1M
water Sucrose
solution
5 10 mL 50mL
distilled 5%
water Albumin
solution
6 10mL 50 mL
your your
choice: choice:

Conclusion Questions
1. Describe what happened in each tube and why. Use the class discussion to help you
answer this question.
1:

2:

3:

4:

5:

‘21-’22 AP Biology Lab Manual 21

2. Which pair(s) that you tested did not have a change in mass? Why?

3. What happened in the “your choice” tube? Explain WHY that happened.

4. Glucose and water are both clear. How could you VISUALLY test for the diffusion of
glucose? Do a little research if you’re stuck (hint: a chemical).

5. If your dialysis tubes were plant cells, what other factor would affect the osmosis that
you witnessed?

‘21-’22 AP Biology Lab Manual 22

Observing Osmosis in Living Cells
First, read the steps in the AP Lab Manual so you know what is done (Procedure 3: Observing
Osmosis in Living Cells). Then, go to YouTube and search “Plant cells in salt water” by
mantismundi (6:17), The link is posted in Google Classroom. We do not have the materials to
do this part of the lab, so you’re going to watch a YouTube video to see what happens, and
then answer the questions based on what you saw. You do not need to watch the whole thing
(it’s all the same for almost 3 minutes)- just enough to see what is going on.

1. Where is the cell membrane in relation to the cell wall? Draw or explain.

2. Which cell organelle in plants controls water concentration inside the cell? (hint: for storage)

3. When the plant cell is placed in salt water, what happens to the cytoplasm and everything
inside the cell?

4. WHY does this happen to the cell? Use proper terminology (hyper/hypo/isotonic etc.)

5. If the cells were placed in an isotonic solution, what would you expect to happen to the
cells?

‘21-’22 AP Biology Lab Manual 23

Design an Experiment
You are going to have a regular potato, sweet potato/yam, Baker’s potato /white potato, and a
red potato (4 potatoes total). You need to find the molar concentration of each potato using
various sucrose solutions. You will have six different colored solutions. The solutions are 0M,
0.2M, 0.4M, 0.6M, 0.8M and 1.0M. However, they will not be labeled, so you will not know
which is which, so you will then use the molarity of the potatoes to determine the concentration
of each solution.
Design an experiment, using osmosis, to identify the concentrations of the various sucrose
solutions. You will then use your data to determine the water potential of the plant tissues.
Hints to guide your setup:
● You need to test all of the potatoes in all of the solutions (you can throw all 3 in the same
beaker together, as long as you have a way to identify which is which).
● You will need to find the percent change in mass for each type of potato.
● What kind of solution (in terms of tonicity) will help you find out the molarity of each
potato?
● Since you won’t know which solutions is 0M, 0.2M, 0.4M, etc., how can you use percent
change in mass to determine this?
● Your graph is going to be different than usual. You will use the graph included at the end
(cut and put in notebook) to record the percent change in mass and help you find the
molarity of each potato.

Come up with some ideas for how to test this lab.

● Create a data table BEFORE doing the lab so you can easily collect your data. Ask to
see Ms. Schwobe’s if you need an idea of how to create it (you need to collect the initial,
final, and % change for each potato (3 total) in all colors of solution (6 colors).
● For the graph, you may work on it together, but each person needs to make their own
(AKA do not make one and then copy it for everyone in the group).

Open a Google Doc/share it with your group and Ms. Schwobe.

Purpose- What is the purpose of this part of the lab?

Safety Information- List (bullet format) safety concerns. If none, write “none.”

Materials- List (bullet format) materials needed.

Hypothesis- Finish this hypothesis:

If a potato is placed in an isotonic solution, then the percent change in the potato’s mass will be
____, because

Procedure- Write, step by step, what you’re doing for this lab. Be specific with measurements
and such! Write as a numbered list. *MAKE SURE TO TAKE THE TEMPERATURE OF
THE ROOM!!!

Data collection- Create the data table for collecting your information.

YOU WILL NOT BE RUNNING THE LAB, YOU ARE SIMPLY TO DESIGN AN EXPERIMENT
THAT WILL ANSWER THE QUESTIONS LISTED ABOVE

‘21-’22 AP Biology Lab Manual 24

 DNA Replication On the Floor
4.3: Describe the mechanism by which genetic information is copied for transmission
between generations.

Setup
1. Obtain 2 pieces of rolled-up paper, each 18 ft long
2. Start with one piece of paper. Across the top, you are going to trace shoe prints. You will
use somebody’s LEFT shoe. (Use the same shoe the entire activity).
3. Choose a purple marker.
4. The heel side of the shoe must be at the LEFT edge of the paper. Trace shoe prints,
heel to toe, down the entire length of the paper from the left edge to the right.
5. In each shoe print, in purple, put in a nitrogenous base- A, T, G, or C

6. Lay the other sheet parallel to the one you just created. Use the same left shoe.
7. Use purple again.
8. The heel side of the shoe must be at the RIGHT edge of the paper. Trace shoe prints,
heel to toe, from the right edge of the paper to the left.
9. MAKE SURE THAT EACH SHOE PRINT IS LINED UP WITH THE ONE ON THE
OTHER STRAND. You should have the exact same number of prints and they need to
be in alignment with each other.

10. In each
shoe
print, in
the same

color, put
the

COMPLEMENTARY BASE to the bases you filled in. This means A goes with T and G
with C!

‘21-’22 AP Biology Lab Manual 25

 11. Tape the LEFT edge of both pieces to the floor, making sure they are still parallel with
each other.
12. Mark your 3’ and 5’ ends. HEELS WILL ALWAYS BE 5’. TOES WILL ALWAYS BE 3’.
13. Starting at the RIGHT end of each paper, roll up each piece. Stop just shy of the taped
ends- leave 3 shoe prints exposed.

Replication
14. Divide up jobs. You need:
a. Helicase person – no marker
b. Primase person – red marker
c. DNA polymerase III person, leading strand – green marker
d. DNA polymerase III person, lagging strand – green marker
e. DNA polymerase I person – black marker
f. Ligase person – yellow marker
g. Nuclease person – brown marker
15. The tape line is going to be your origin of replication. Your helicase person will “unzip”
your DNA strands by unrolling the right ends as the DNA is replicated. Start by exposing
6 footprints.
16. The Primase person needs a RED marker and a left shoe (continue to use the same
original shoe for the whole modeling). On the leading strand (bottom, 3-5), they should
trace and “lay down” some RNA nucleotides (the RNA Primer).
17. Start on the left side by the tape.
18. Trace 3 shoe prints in red directly above the first 3 prints on the paper. Toe matches
with heel, heel with toe.

19. Fill in the appropriate base pair to go with the DNA. REMEMBER!!! U’s now replace
T’s! So A pairs with U, T with A, and C with G.
20. DNA polymerase III person, leading strand, needs a GREEN marker and a left shoe.
21. Now that you have a 3’ end to work with, you can lay down DNA nucleotides. Add 3
green shoe prints.
22. Fill in the bases as they pair with the other strand.

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23. Now we’re going to synthesize the lagging strand. Remember, this happens
simultaneously in real DNA replication, but to make a point, we’re doing this in pieces.
24. First, PRIMASE needs to add a primer with a RED marker and a left shoe, just like
before.
25. Start by the replication fork (where the paper is rolled up).
26. Trace 3 shoe prints in red directly above the first 3 prints on the paper. Toe matches
with heel, heel with toe.
27. Fill in the base pairs with RNA nucleotides.

28. DNA polymerase person III, lagging strand, needs a GREEN marker and a left shoe.
29. Now that you have a 3’ end to work with, you can lay down DNA nucleotides. Add 3
green shoe prints.
30. Fill in the bases as they pair with the other strand.
31. Helicase person: expose 6 more footprints.
32. DNA polymerase person III, leading strand: you don’t need to wait for primase. Keep
making more DNA! All you need is a 3’ end to work with, and you still have that (the toe
of the last shoe). So keep on keepin’ on!
33. DNA polymerase person III, lagging strand- hold your horses! Oh no! More DNA is
exposed. You need to “jump” ahead to replicate this new portion. But first…you need a
primer.
34. Primase person, just like before, make a 3-foot primer. THEN, DNA polymerase person
III, lagging strand- go ahead and finish up your next segment.
35. Helicase person: expose the last footprints. Other proteins…you know what to do!
36. DNA polymerase I person- it’s your turn! You need to “remove” the RNA primers and
replace them with DNA. To represent doing this, take a BLACK marker and trace over
the nucleotides of the primer. Remember to change any U’s to T’s!
37. Ligase person- your job is to “glue” segments together. Anywhere you see an RNA
primer next to a DNA strand, please use a YELLOW marker and color in the space
between the toe of the primer and the heel of the DNA strand. This means that the
leading strand will have one yellow line, and the lagging strand will have three (one for
each Okazaki fragment).

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 38. Nuclease person- your job is to check for errors. Skim both strands of DNA and make
sure A’s are paired with T’s and G’s with C’s. If not, please fix the letters using a
BROWN marker.

You should now have two identical copies of DNA. Yay!

1. Explain HOW the synthesis of the leading strand differs from that of the lagging strand.

2. Explain WHY the synthesis of the leading strand differs from that of the lagging strand.

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 Gel Electrophoresis Lab
5.3: Simplify the different types/procedures of biotechnology, and explain the importance
of these scientific tools.

Materials:
● Sterile pipette tips
● Micropipette
● 1X Buffer
● Electrophoresis chamber
● Agarose gel
● Power Supply
● Gel comb
● Loading dye samples

Directions:
1. Put on rubber gloves. (Steps 2-3 are already done for you)
2. Place the tray in the chamber oriented correctly (negative sign by the negative node,
positive sign by the positive node).
3. Fill the gel electrophoresis chamber with 1X TBE (Tris-Borate-EDTA) buffer to a level
that just covers the surface of the gel.
4. The gel is now ready to load. Ms.Schwobe will come around with each dye in
chronological order.
5. You will load 20 microliters into each well. Be sure to use a new micropipette tip each
time, and discard the old ones in the trash.
6. Load the dye samples into the wells from left to right in the order listed below.
1 2 3 4 5 6 7 8

Bromophenol Methyl Ponceau Xylene Pyronin Y Unknown Unknown Unknown

blue orange G cyanol #1 #2 #3

7. Once all the samples have been loaded, place the lid on the electrophoresis chamber.
8. Make sure the lid is oriented correctly.
9. Connect the electrical cords to the power supply, and turn on the power supply.
10. Turn to the correct voltage, and run the gel until the band in lane 3 is 0.5 cm from the
end of the gel.

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 11. Fill in the results table below:
Lane Sample Number of Direction of Migration Migration
Bands (Positive or Negative) Distance
(cm)

1 Bromophenol Blue

2 Methyl Orange

3 Ponceau G

4 Xylene Cyanol

5 Pyronin Y

6 Unknown #1

7 Unknown #2

8 Unknown #3

12. Turn off the power, and disconnect the cords from the power supply.
13. Remove the top from the electrophoresis chamber.
14. Carefully remove the casting tray, and slide the gel into the plastic tray.

Questions:
1. Based on the direction of migration distance, and the appearance of your gel, what dye
component(s) were present in each of the unknown dye mixtures?
a. Unknown #1:

b. Unknown #2:

c. Unknown #3:

2. Which dye molecules traveled the farthest through the gel?

3. Which dye molecules traveled the shortest?

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4. What properties might affect migration distance?

5. What was the charge of the dye molecules that migrated toward the positive electrode
and of the dye molecules that migrated toward the negative electrode?

6. Why is electrical current necessary for separating molecules by gel electrophoresis?

7. Why is the porous matrix of agarose gels an essential component of molecules

separation by gel electrophoresis?

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 Pill Bug Behavior Choices
Overview
Pill bugs, also known as roly polies, are actually terrestrial crustaceans. Pill bugs (Armadillidium
vulgare) have gills for respiratory organs like their aquatic cousins. Because pill bugs have gills,
their habitat is restricted to damp places. It is common to find pill bugs under wood or leaf litter,
on the bottom of flower pots, or in damp corners. They burrow into the ground and feed on leaf
litter or pieces of fruit or vegetables like apples, potatoes, and carrots. An adult female can carry
up to 200 eggs in a brood pouch on the ventral side of her thorax. The young hatch in the pouch
and stay there for about 3 weeks. Young pill bugs are smaller in size and paler in color than
adults. The young will molt 4 to 5 times as they grow.

Essential Question
Do environmental conditions affect animal behavior?

Investigation Objectives
1. Investigate how pill bugs respond to humidity levels in their environment.
2. Identify 2 other environmental factors that influence pill bug behavior.

Materials
● 1 cup of 10 pill bugs
● 2 pieces of filter paper or paper
● towel
● 1 cup of water
● 1 choice chamber with 2
● chambers
● 1 plastic spoon
● 1 dropper
● 1 stopwatch or timer

Procedure
1. Place a clean sheet of filter paper or paper towel disk into each side of the choice
chamber.
2. Using the dropping pipet, dampen the filter paper on 1 side of the chamber. Make sure
the paper absorbs all excess water.
3. Use a plastic spoon to transfer 5 pill bugs to each side of the chamber.
4. Put the lids on the chamber.
5. Count and record on the data table the number of pill bugs on each side of the chamber
every 30 second

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Data Tables

Time # on damp # on dry Time # on damp # on dry

(min:sec) side side (min:sec) side side
0:00 5:30
0:30 6:00
1:00 6:30
1:30 7:00
2:00 7:30
2:30 8:00
3:00 8:30
3:30 9:00
4:00 9:30
4:30 10:00
5:00

Analysis and Discussion

1. Graph your data for the damp side and the dry side of the chamber on the same axes.
Title the graph and include the following information:
a. The independent variable is ________________
b. The dependent variable is __________________
c. Plot the independent variable on the x-axis and the dependent variable on the y-
axis.

2. Based on your graph and the

introductory material, explain pill bug
behavior with respect to humidity.

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3. Explain how the pill bug response to humidity may be advantageous.

4. Identify 2 more environmental factors that may affect pill bug behavior.

5. Design an experiment to test 1 of the environmental factors that may affect pill bug
behavior

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 pGLO Transformation Lab

Lesson 1 Introduction to Transformation

In this lab you will perform a procedure known as genetic transformation. Remember
that a gene is a piece of DNA which provides the instructions for making (codes for) a protein.
This protein gives an organism a particular trait. Genetic transformation literally means “change
caused by genes,” and involves the insertion of a gene into an organism in order to change the
organism’s trait. Genetic transformation is used in many areas of biotechnology. In agriculture,
genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed
into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them
to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated
by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of
the defective gene that causes the disease.
You will use a procedure to transform bacteria with a gene that codes for Green
Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish
Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the
dark. Following the transformation procedure, the bacteria express their newly acquired jellyfish
gene and produce the fluorescent protein, which causes them to glow a brilliant green color
under ultraviolet light.
In this activity, you will learn about the process of moving genes from one organism to
another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally
contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually
contains genes for one or more traits that may be beneficial to bacterial survival. In nature,
bacteria can transfer plasmids back and forth allowing them to share these beneficial genes.
This natural mechanism allows bacteria to adapt to new environments. The recent occurrence
of bacterial resistance to antibiotics is due to the transmission of plasmids.
Bio-Rad’s unique pGLO plasmid encodes the gene for GFP and a gene for resistance to
the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which can be
used to control expression of the fluorescent protein in transformed cells. The gene for GFP can
be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium.
Selection for cells that have been transformed with pGLO DNA is accomplished by growth on
ampicillin plates. Transformed cells will appear white (wild-type phenotype) on plates not
containing arabinose, and fluorescent green under UV light when arabinose is included in the
nutrient agar medium.

You will be provided with the tools and a protocol for performing genetic transformation.
Your task will be to:
1. Do the genetic transformation.
2. Determine the degree of success in your efforts to genetically alter an organism.

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Lesson 1 Focus Questions

There are many considerations that need to be thought through in the process of
planning a scientific laboratory investigation. Below are a few for you to ponder as you take on
the challenge of doing a genetic transformation.
Since scientific laboratory investigations are designed to get information about a
question, our first step might be to formulate a question for this investigation.
Consideration 1: Can I Genetically Transform an Organism? Which Organism?
1. To genetically transform an entire organism, you must insert the new gene into every cell
in the organism. Which organism is better suited for total genetic transformation— one
composed of many cells, or one composed of a single cell?

2. Scientists often want to know if the genetically transformed organism can pass its new
traits on to its offspring and future generations. To get this information, which would be a
better candidate for your investigation, an organism in which each new generation
develops and reproduces quickly, or one which does this more slowly?

3. Safety is another important consideration in choosing an experimental organism. What

traits or characteristics should the organism have (or not have) to be sure it will not harm
you or the environment?

4. Based on the above considerations, which would be the best choice for a genetic
transformation: a bacterium, earthworm, fish, or mouse? Describe your reasoning.

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Consideration 2: How Can I Tell if Cells Have Been Genetically Transformed?
Recall that the goal of genetic transformation is to change an organism’s traits, also
known as their phenotype. Before any change in the phenotype of an organism can be
detected, a thorough examination of its natural (pre-transformation) phenotype must be made.
Look at the colonies of E. coli on your starter plates. List all observable traits or characteristics
that can be described:

The following pre-transformation observations of E. coli might provide baseline data to make
reference to when attempting to determine if any genetic transformation has occurred.

1. Number of colonies

2. Size of :
a. the largest colony

b. the smallest colony

c. the majority of colonies

3. Color of the colonies

4. Distribution of the colonies on the plate

5. Visible appearance when viewed with ultraviolet (UV) light

6. The ability of the cells to live and reproduce in the presence of an antibiotic such as
ampicillin

7. Describe how you could use two LB/agar plates, some E. coli and some ampicillin to
determine how E. coli cells are affected by ampicillin.

8. What would you expect your experimental results to indicate about the effect of
ampicillin on the E. coli cells?

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Consideration 3: The Genes
Genetic transformation involves the insertion of some new DNA into the E. coli cells. In
addition to one large chromosome, bacteria often contain one or more small circular pieces of
DNA called plasmids. Plasmid DNA usually contains genes for more than one trait. Scientists
use a process called genetic engineering to insert genes coding for new traits into a plasmid. In
this case, the pGLO plasmid has been genetically engineered to carry the GFP gene which
codes for the green fluorescent protein, GFP, and a gene (bla) that codes for a protein that
gives the bacteria resistance to an antibiotic. The genetically engineered plasmid can then be
used to genetically transform bacteria to give them this new trait.

Consideration 4: The Act of Transformation

This transformation procedure involves three main steps. These steps are intended to
introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to
express their newly acquired genes.

To move the pGLO plasmid DNA through the cell membrane you will:
1. Use a transformation solution containing CaCl2 (calcium chloride).
2. Carry out a procedure referred to as heat shock.
For transformed cells to grow in the presence of ampicillin you must:
1. Provide them with nutrients and a short incubation period to begin expressing their
newly acquired genes.

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Lesson 2 Transformation Laboratory

Your workstation: Materials and supplies that should be present at your workstation prior to
beginning this lab are listed below.

Common workstation. A list of materials, supplies, and equipment that should be present at a
common location to be accessed by your team is also listed below

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Transformation Procedure:
1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with
your group’s name. Place them in the foam tube rack.

2. Open the tubes and, using a

sterile transfer pipette, transfer
250 µl of transformation solution
(CaCl2) into each tube.

3. Place the tubes on ice.

4. Use a sterile loop to pick up 2–4

large colonies of bacteria from
your starter plate. Select starter
colonies that are "fat" (ie: 1–2 mm
in diameter). It is important to take
individual colonies (not a swab of
bacteria from the dense portion of
the plate), since the bacteria must
be actively growing to achieve high
transformation efficiency. Choose
only bacterial colonies that are
uniformly circular with smooth
edges. Pick up the +pGLO tube
and immerse the loop into the transformation solution at the bottom of the tube. Spin the
loop between your index finger and thumb until the entire colony is dispersed in the
transformation solution (with no floating chunks). Place the tube back in the tube rack in
the ice. Using a new sterile loop, repeat for the -pGLO tube.

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5. Examine the pGLO DNA solution with the UV lamp. Note your observations. Immerse a
new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There
should be a film of plasmid solution across the ring. This is similar to seeing a soapy film
across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the
+pGLO tube. Optionally, pipet 10 µl of pGLO plasmid into the +pGLO tube & mix. Do
not add plasmid DNA to the -pGLO tube. Close both the +pGLO and -pGLO tubes and
return them to the rack on ice.

6. Incubate the tubes on ice for 10 min. Make sure to push the tubes all the way down in
the rack so the bottom of the tubes stick out and make contact with the ice.
7. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom
(not the lid) as follows:
a. Label one LB/amp plate: + pGLO
b. Label the LB/amp/ara plate: + pGLO
c. Label the other LB/amp plate: - pGLO
d. Label the LB plate: - pGLO

8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO
tubes into the water bath, set at 42°C, for exactly 50 sec. Make sure to push the tubes
all the way down in the rack so the bottom of the tubes stick out and make contact with
the warm water. Double-check the temperature of the water bath with two thermometers
to ensure accuracy.

When the 50 sec are done, place both tubes back on ice. For the best transformation
results, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid.
Incubate tubes on ice for 2 min.

9. Remove the rack containing the

tubes from the ice and place on the

‘21-’22 AP Biology Lab Manual 41

 bench top. Open a tube
and, using a new sterile
pipet, add 250 µl of LB
nutrient broth to the tube
and reclose it. Repeat with
a new sterile pipet for the
other tube. Incubate the
tubes for 10 min at room
temperature.

10. Gently flick the closed

tubes with your finger to
mix and resuspend the
bacteria. Using a new sterile pipet for each tube, pipet 100 µl of the transformation and
control suspensions onto the appropriate nutrient agar plates.

11. Use a new sterile loop for each plate. Spread the suspensions evenly around the
surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop

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 back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR.
Uncover one plate at a time and re-cover immediately after spreading the suspension of
cells. This will minimize contamination.

12. Stack up your plates and tape them together. Put your group name and class period on
the bottom of the stack and place the stack of plates upside down in the 37°C incubator
until the next day. The plates are inverted to prevent condensation on the lid which may
drip onto the culture and interfere with your results.

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Lesson 2 Review Questions
1. On which of the plates would you expect to find bacteria most like the original non-
transformed E. coli colonies you initially observed? Explain your predictions

2. If there are any genetically transformed bacterial cells, on which plate(s) would they
most likely be located? Explain your predictions.

3. Which plates should be compared to determine if any genetic transformation has

occurred? Why?

4. What is meant by a control plate? What purpose does a control serve?

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Lesson 3 Data Collection and Analysis

A. Data Collection
Observe the results you obtained from the transformation lab under normal room
lighting. Then turn out the lights and hold the ultraviolet light over the plates. Alternatively the
protocol can incorporate digital documentation of the plates with Vernier’s Blue Digital
BioImaging System

1. Carefully observe and draw what you see on each of the four plates. Put your drawings
in the data table below. Record your data to allow you to compare observations of the “+
pGLO” cells with your observations for the non-transformed E. coli. Write down the
following observations for each plate.

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 2. How much bacterial growth do you see on each plate, relatively speaking?

3. What color are the bacteria?

4. How many bacterial colonies are on each plate (count the spots you see).

B. Analysis of Results
The goal of data analysis for this investigation is to determine if genetic transformation
has occurred.
1. Which of the traits that you originally observed for E. coli did not seem to become
altered? In the space below list these untransformed traits and how you arrived at this
analysis for each trait listed.

Original trait Analysis of observations

2. Of the E. coli traits you originally noted, which seem now to be significantly different after
performing the transformation procedure? List those traits below and describe the
changes that you observed.

New trait Observed change

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 3. If the genetically transformed cells have acquired the ability to live in the presence of the
antibiotic ampicillin, then what might be inferred about the other genes on the plasmid
that you used in your transformation procedure?

4. From the results that you obtained, how could you prove that the changes that occurred
were due to the procedure that you performed?

Lesson 3 Review Questions

What’s Glowing?
If a fluorescent green color is observed in the E. coli colonies then a new question might
well be raised, “What are the two possible sources of fluorescence within the colonies when
exposed to UV light?”

Explain:

1. Recall what you observed when you shined the UV light onto a sample of original pGLO
plasmid DNA and describe your observations.

2. Which of the two possible sources of the fluorescence can now be eliminated?

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 3. What does this observation indicate about the source of the fluorescence?

4. Describe the evidence that indicates whether your attempt at performing a genetic
transformation was successful or not successful.

The Interaction between Genes and Environment

Look again at your four plates. Do you observe some E. coli growing on the LB plate that
does not contain ampicillin or arabinose?

1. From your results, can you tell if these bacteria are ampicillin resistant by looking at
them on the LB plate? Explain your answer.

2. How would you change the bacteria’s environment—the plate they are growing on—to
best tell if they are ampicillin resistant?

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3. Very often an organism’s traits are caused by a combination of its genes and its
environment. Think about the green color you saw in the genetically transformed
bacteria:
a. What two factors must be present in the bacteria’s environment for you to see the
green color? (Hint: one factor is in the plate and the other factor is in how you
look at the bacteria).

b. What do you think each of the two environmental factors you listed above are
doing to cause the genetically transformed bacteria to turn green?

c. What advantage would there be for an organism to be able to turn on or off

particular genes in response to certain conditions?

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