BIOL 1100 Laboraory Manual August 2019

Biology 1100 Lab Manual
3rd Edition
Written and Compiled by
Nathan Lanning
Susan Cohen
Elizabeth Torres
Valerie Wong
Kirsten Fisher
Department of Biological Sciences

California State University, Los Angeles
2019
Table of Contents
Exercise 1: Micropipeting Basics .............................................................................................................. 3

Exercise 2: Introduction to Microscopes ................................................................................................. 8
Exercise 3: Diffusion and Osmosis ......................................................................................................... 17
Exercise 4: Yeast Respiration and Fermentation .................................................................................. 29
Exercise 5: Bacterial Growth Analysis Part A ...................................................................................... 36
Exercise 6: Bacterial Growth Analysis Part B ...................................................................................... 40
Exercise 7: Using Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability Part A ......... 44
Exercise 8: Using Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability Part B ......... 50
Exercise 9: Using Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability Part C ......... 56
Exercise 10: Bioinformatics .................................................................................................................... 59
Exercise 1:
Micropipetting Basics
Laboratory Objectives
After completing this laboratory topic, you should be able to:

1. Accurately pipet different microliter volumes using a micropipette.
2. Micropipette solutions of different viscosities.
3. Perform conversions between volumetric units of measure and express values in
scientific notation and decimals.
Introduction
Over the past several decades, advances in biotechnology have influenced many changes in
experimental techniques and methods, including the volume of reagents and biological
samples used. Depending upon the procedure being performed, biotechnology experiments
can utilize a variety of volumes of biological samples and reagents, ranging from several
hundreds of liters to very small microliter (µl) volumes.
Pipeting is a critically important technique in life science experiments to ensure accurate

experimental results. In typical biotechnology experiments, biologicals and reagents such as
DNA, enzymes and buffers are transferred (by pipetting) into small microcentrifuge tubes
(Figure 1.1), which serve as reaction vessels. For these types of reactions, microliter
volumes are typically used. There are 1,000 microliters in 1 milliliter of a solution. To put it in
perspective, a 50 microliter sample is approximately equal in size to a single raindrop. A
raindrop-sized sample is relatively large compared to experimental samples, which often are
10 to 50 microliters in volume.
Figure 1.1. Microcentrifuge tubes.
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To measure microliter volumes, a special instrument called a micropipette is used (Figure 1.2).
The variable volume micropipette is the preferred instrument for delivering accurate,
reproducible volumes of sample. These instruments are manufactured to deliver samples in
various ranges (e.g., 0.5-10 µl, 5-50 µl, 200-1000 µl, etc.) and usually can be adjusted in one
microliter increments or less. Typically, these instruments have an ejector button for releasing
the tip after sample delivery. Variable volume micropipettes can also be multi-channeled
(Figure 1.3), designed to uniformly deliver several samples at the same time. However, for this
experiment, only one sample will be delivered at a time.
Figure 1.2. Single-channel variable Figure 1.3. Multi-channel variable

volume micropipet. volume micropipet.
Materials
Dyes (Red, Blue, Yellow)

Glycerol
Alcohol
Buffer
Microcentrifuge tubes
Micropipettes of variable volumetric calibrations
Micropipette tips
Small container for discarding used tips
Gloves
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Procedure
Measuring Small Volumes with Micropipettes
Prior to beginning the protocol below, it is essential that you understand which micropipette
to use for a given volume. Choosing the wrong micropipette will result in you dispensing an
incorrect volume or the micropipette becoming damaged. Typically, the top of each
micropipette will be labeled with the volume range for which it is designed. BIOL 1100 has
the following micropipettes available:
Typical Micropipette Labels and Ranges

Volume Example
Name Label Range Example Volume Display
P10 1-10 1μl - 10μl 5μl 0 5 0
P20 2-20 2μl - 20μl 11μl 1 1 0
P100 10-100 10μl - 100μl 25μl 0 2 5
P200 20-200 20μl - 200μl 175μl 1 7 5
P1000 100-1000 100μl - 1000μl 550μl 0 5 5
For this exercise, make sure you choose the appropriate micropipette for each measurement
based on the volume of liquid that you are measuring. Never adjust the micropipette over or
under the calibrated volume range as this will damage the pipette.
Steps 1-4 in the protocol below correspond to the numbered steps in the instructional
drawing (Figure 1.4). You should always wear gloves to protect yourself from any reagents
that have come into contact with the micropipette and to protect your experiment from
contamination from your skin. Never handle microcentrifuge tubes without gloves if the
experiment requires a sterile approach.
1. Set the micropipette to the appropriate volume and place a clean tip on the
micropipette. This is done by gently and carefully pressing the tip of the micropipette
into a box of clean tips. Once the tip is attached to the micropipette, press the top
button down to the first stop and hold it in place while placing the tip into the sample
tube (Step 1).
2. Once the tip is immersed in the sample, release the button slowly to draw sample into
the tip (Step 2). If you release the button too quickly, the liquid will splash into the top
portion of the tip. Your volume will be inaccurate.
3. Deliver the sample by pressing the button to the first stop — then empty the entire
contents of the tip by pressing to the second stop (Step 3).
Note: After delivering the sample, do not release the top button until the tip is out of the
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tube or vessel to which the sample is delivered.
4. Press the ejector button to discard the tip. Obtain a new clean tip for the next sample.
It is important to change tips so that you do not contaminate reagents.
Figure 1.4. Sample delivery with micropipets.
Micropipetting Using a Variable Volume Micropipette
In this activity you will:
1. As a group, use micropipettes to first prepare seven different dye mixtures in separate
microcentrifuge tubes (Table 1). Each group member should be responsible for
preparing at least one of the dye mixtures. Some of the group members will be
required to prepare two dye mixtures.
2. Next, each member of the group will choose one of the mixtures that you prepared (it
does not matter which mixture each group member chooses) and perform a
quantitative transfer of the volume from that tube to a new empty tube in order to
assess the accuracy of the volume of the assembled solution.
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Table 1. Dye Mixtures to be prepared.
Protocol:
1) Obtain seven microcentrifuge tubes. Label the tubes 1 to 7 with a marker.
2) To the tube labeled #1, add 5 µl of red dye and 40 µl of buffer for a total volume
of 45 µl. Repeat this process for the different solutions according to Table 1 for
tubes 2-7. Each student must complete at least one tube. Visually compare the
volumes of each tube. Do the volumes appear approximately equivalent?
3) Each group member should now obtain a single new tube and label the tube with
their initials. Then, each group member should select one of the tubes that were
just prepared with 45 μl of dye mixture (it does not matter which tube each group
member selects).
4) Using either a P20 or P10 micropipette, each group member will now transfer the
contents from the dye mixture tube that you selected into the single new tube
labeled with your initials as follows:
a. Transfer 10uL from the dye mixture tube into the new tube.
b. Repeat step 4a until less than 10uL remains in the dye mixture tube.
c. Adjust the volume on the micropipette to match as closely as possible the
volume remaining in the dye mixture tube, and then transfer it to the new
tube with your initials.
5) Add the volumes you transferred from the dye mixture tube to the new tube for
steps 4a-c (e.g., 10uL + 10uL + … + … = ?).
6) Record this final calculated volume in your laboratory notebook. Please also
record your final calculated volume in the data table that your lab instructor will
pass around the room.
Lab Instructors: hand the completed data table to your lab coordinator
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Exercise 2:
Introduction to Microscopes
Exercise compiled and adapted from resources:
https://www.biologycorner.com/worksheets/e-lab.html
https://www.biologycorner.com/worksheets/microscope-investigation.html
https://www.biologycorner.com/worksheets/microscope-advanced.html
Labeled Image of Leica CME: http://medicine2.keele.ac.uk/microscope/Microscope_StepByStep.html
1. Compare and contrast different types of microscopes.

2. Learn how to identify the parts of the microscope and confidently use the microscope.
3. Learn the proper use of a microscope and be able to focus using each of the lenses.
4. Create a wet mount and stain a slide for easier viewing.
5. Learn how to estimate the size of microscopic objects.
Materials
Glass slides
Cover slips
Cotton balls for fibers
Forceps for removing fibers from cotton balls
Methylene blue or iodine in a dropper bottle
Lens Paper
Lens cleaner solution (optional)
Small flat ruler
Stereoscopes for demonstration purposes with any type of specimen for view.
Per group of two students:

1 compound microscope
Slides to observe: “e slide”, Colored thread slide, Paramecium slide, Cork slide
Transparent ruler
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Introduction
What are the Different Types of Microscopes?

One of the most important tools that a biologist uses is the microscope. In this course, you will use two
types of light microscopes: the compound light microscope with magnifications ranging from 40x to
400x and the stereomicroscope with magnifications ranging from 7x to 20x. Each of these
microscopes focuses light through two lenses in order to magnify objects in the viewing field.
Stereoscopes are also called dissecting microscopes because they allow large specimens to be
viewed closely. Most are binocular (two eyepieces).
In addition to those microscopes, biologists can also use scanning electron microscopes (SEM) and
transmission electron microscopes (TEM). These tools use electrons instead of light to create an image
of the specimen. Generally, an SEM focuses on the surface of the specimen, creating a 3-dimensional
view. The TEM beams the electrons through the specimen, revealing internal structures. The electron
microscopes have greater magnifications than the light microscopes but cannot be used to view living
specimens. Most beginning students will not have access to electron microscopes.
Each type of microscope has advantages and disadvantages for viewing specimens and each creates
unique views. The images below show plants as seen under three types of microscopes. The SEM
shows the surface of the leaf with a close-up of a trichome, the TEM shows the chloroplast of a leaf
cell, and the light microscope shows the individual cells within the leaf. A biologist will choose a
microscope based on what observations need to be made.
Fluorescence Microscope
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In order to see structures, some specimens may need to be coated or stained. Fluorescent dyes are
used to stain and view specific molecules, improving the ability to study cells from prepared slides or in
cultures living inside of petri dishes or culture plates. Compound microscopes equipped with lasers and
filters allow scientists to stimulate the fluorescent dyes and view or image the structures. Fluorescence
microscopy has become a standard approach in life science research.
Each microscope can be defined by its magnification and its resolving power (resolution.) Resolution
refers to the microscope's ability to distinguish two closely spaced objects. The resolving power of the
human eye is about 0.1 millimeters, meaning that small objects that are closer than that will be
perceived by the eye as a single object.
Getting to Know the Microscope
Review the parts of the microscope as they are illustrated below. You may have used a microscope in
the past and have some familiarity. Your microscope may vary slightly from the image, but most
compound light microscopes have the same general design.
Before working with the

microscope, watch video
by scanning QR code or
at the following link:
https://www.youtube.com/
watch?v=RJe577AQqvA
A. General Use
- Carry the microscope with one hand on the base and the other on the arm.
- Use lens paper to clean the lenses, other types of paper can scratch the glass. Ask your lab instructor
for help if you have any questions about properly cleaning the eyepiece or objective lenses.
- Do not place microscope near the edge of table. Keep area clear of backpacks to prevent accidents.
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- Do not drag the microscope to move it on the table, lift it with two hands.
- Always wrap cords when finished (do not leave plugged in) and cover microscopes.
B. Magnification
Familiarize yourself with the objectives. What magnification is written on each of them? Magnification is
written with a number followed by an X, for example, 10X means that the image you see is 10 times
bigger than actual size.
Your microscope has four objectives. The magnification written on the ocular lens (eyepiece) = 10X ,
the Scanning Objective = 4X, Low Power =10X, High Power = 40X, and Oil Immersion Objective =
100X.
The oil immersion objective only works with a small drop of oil placed between the cover slip and the
lens of the objective. This should be done with extreme care and oil removed with lens paper after use.
It is extremely important that oil does not get on the other objectives or on the other parts of the
microscope. Only use oil and oil immersion objective if directed to do so by your lab instructor.
Determine the total magnification achieved by each objective. Write your answer in the blank
boxes of table.
**The total magnification is calculated by multiplying the ocular and the objective. **
Ocular Lens Scanning Objective Low Power High Power Oil Immersion Objective
10X 4X Objective Objective 100X
10X 40X
Total = 10X Total = Total = Total = Total =
D. Focusing the Microscope (4 Basic Steps – details of how to correctly place slide on stage in
the video)
1) Use the scanning objective (4X) to scan the slide to find a good viewing spot.
2) Use the coarse adjustment knob to bring object into focus
3) Switch to low power (10X) and use the coarse knob to bring it back into focus. You may also need to
re-center the specimen.
4) Switch to high power (40X), At this point, ONLY use the fine adjustment knob to bring the object into
focus.
E. How Can You Adjust the Light
Examine the diaphragm, determine how to change the amount of light that passes through your
viewing field. Diaphragms on microscopes can be different depending on the model. Some may be
shaped like wheels with several settings. You can also adjust the light intensity with the dial on the base
of the microscope.
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In some cases, a dim light might be better for viewing cells. You may want to experiment with the
diaphragm to find the best setting for your specimen.
Pre Lab Questions
1. What is the difference between magnification and resolving power?
2. Which objective do you use to locate specimens on your slide? _________________________

3. Which adjustment knob is used when focusing the high power objective? ____________________
4. Why might you want to adjust the diaphragm?
Part 1: How Can You Focus the Microscope Using the E slide?
Adjust the nosepiece of the microscope so that the scanning lens (4x) is in place. Place the e-slide
on the stage so that you can visible see the letter "e" is over the opening where light will enter.
While looking into the ocular lens, use the coarse adjustment knob to bring the "e' into focus.
Carefully re-center the e on the slide and switch to the low

power objective (10x). The specimen will be out of focus and it
may have shifted slightly. Use the coarse adjustment
knob again to bring the "e" back into focus.
Carefully re-center the "e" again and switch to the high

power objective (40x). At this point, DO NOT USE the
coarse focus knob!!
Use the fine adjustment knob only to bring the specimen

back into focus. At this point, you will not see the letter "e," you
will only see the edge, or the ink.
Draw the letter “e” to scale. The circles below represent
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your viewing field. The e should take up as much space in the drawing as it does in your viewing field
while you're looking at it.
Focus your e slide at each magnification. What happens to the working distance as you move from
low to medium to high power? Using this information, discuss with your lab partner why you should
NOT use the coarse adjust when using the high power objective.
Part 1 Analysis:
1. You were asked to draw your object to scale, using the viewing field circles. What does that mean?
2. What happens to the working distance as you change objectives?
3. What happens to the location of the object on the slide when you switch objectives?
Part 2: What is the Field of View and How Can It Be Measured?
The circle of light seen through the eyepiece is the field of view. The field of view varies
by microscope and by the size of the lens, a number that is usually written on the side of
the lens. You will be measuring the field of view for your own microscope, which may be
different than other microscopes in the room.
Use a ruler to determine the width of the viewing field under the scanning objective.
Position the ruler so that the millimeter marks are visible in your viewing field.
Count the number of millimeters; you may need to estimate the fraction of millimeter
involved in the last section. Repeat this procedure to estimate the medium power field of
view. There are 1000 micrometers ( µ ) in a millimeter.
What is your estimate of the length (diameter) of your viewing field in millimeters:
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Scanning (40x) _______ Low Power (100x) ________
What is your estimate of the length (diameter) of your viewing field in micrometers
Scanning (40x) _______ Low Power (100x) ________
You cannot use this method to determine the diameter under high power (try switching objectives, you won't
be able to even see the ruler). Instead you can use a mathematical proportion method to determine the diameter
under high power. Show your work below with your estimate of the length (in micrometers) of your high power
field of view.
High Power Field of View = _______
9. Sketch a paramecium and a cork slide at 400x, drawing each to scale with attention to detail.
Length of paramecium = ____________ Length & Width of single cork cell = ____________
Part 2 Analysis:
1. Check that each section of this page is complete. ✅
2. Explain why you cannot use the ruler technique to estimate specimens at high power.
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Part 3: Working with Specimens
A. Making a Wet Mount of a Slide
Place a drop of water on the slide. Gather a few strands of cotton using forceps and place them on the drop.
Place the coverslip at about an angle with one edge touching the water drop and then gently let go.
Performed correctly the cover slip will perfectly fall over the specimen and will not have air bubbles. If the cover
slip does not sit flat, then you have too much fiber on the slide. If this is the case, start over.
Draw the specimen as it appears in your viewing field under scanning, low and high power. Draw the strands
to scale. Save the slide for part B below.
B. Staining a Specimen
Biologists learn to stain specimens that have already been created. You will not remake the slide, but use a
procedure to draw a stain under the coverslip.
Place one drop of stain (methylene blue or iodine) on

the edge of the coverslip you made with the cotton.
Place the flat edge of a piece of paper towel on the
opposite side of the coverslip. The paper towel will draw the
stain across the slide due to the cohesion property of
water.
View the slide again, note the appearance of the cotton
fibers now compared to the view without the stain.
C. Viewing Large Specimens and Dissecting

Scopes
Light microscopes are only useful for viewing small thin specimens. Stereoscopes present a larger field of
viewing and handle depth much better than the light microscope. The drawback of the stereoscope is that it does
not have a high magnification. Examine one of the stereoscopes in the room which will have a variety of
specimens to view.
Name/Description of specimen(s) viewed: _________________________________________________
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D. Depth Perception
Obtain a slide with three different colored threads on it. View the slide under scanning and then low
power. You should note that you could only focus on one colored thread at one time. Figure out which
thread is on top by lowering your stage all the way, then slowly raising it until the thread comes into
focus. The first thread to come into focus is the one on top. Make sure to pay attention to the working
distance and not crush the slide (should not be a problem at scanning or low power).
Which color thread is on top? _____________ middle? ______________ bottom?

_____________
Part 3 Analysis:
1. Describe the difference when you viewed unstained versus stained cotton fibers.
2. What are the advantages of using a stereoscope? What are the disadvantages?
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Exercise 3:
Diffusion and Osmosis
1. Understand the relationship between molecular size or charge and the rate of
diffusion across a semi-permeable membrane
2. Understand the movement of water in the process of osmosis and the relationship of
this movement to the concentration of osmotically active substances
3. Understand the concept of osmotic pressure
4. Describe osmotic behavior in plant cells, and understand the relationship between the
osmolarity (solute concentration) of the surrounding solution and this behavior
5. Understand that each cell type has a unique osmolarity, and be able to quantitatively
estimate the osmolarity of plant (potato tuber) cells experimentally with the use of
solutions of varying solute concentrations
Introduction
Maintenance of the steady state of a cell is achieved only through regulated

movement of materials through the cytoplasm, across organelle membranes and across the
plasma membrane. This regulated movement facilitates communication within the cell and
between the cytoplasm and the external environment of the cell. Cells are composed of
aqueous solutions. They contain water, which is the solvent, or dissolving agent, and
numerous organic and inorganic molecules, which are the solutes, or dissolved substances.
Organelle membranes and the plasma membrane are selectively permeable, allowing water
to freely pass through but regulating the movement of solutes.
The cell actively moves some dissolved substances across membranes, expending
adenosine triphosphate (ATP) (biological energy) to accomplish the movement. Other
substances move passively without expenditure of ATP from the cell, but only if the cell
membrane is permeable to those substances. Water and selected solutes move passively
through the cell and cell membranes by diffusion, a physical process in which molecules
move from an area where they are in high concentration to one where their concentration is
lower. The energy driving diffusion comes only from the intrinsic kinetic energy in all atoms
and molecules. If nothing hinders the movement, a solute will diffuse until it reaches
equilibrium (i.e., its concentration is the same on both sides of the membrane).
Osmosis is a type of diffusion, the diffusion of water through a selectively permeable
membrane from a region where it is highly concentrated to a region where its concentration
is lower. The difference in concentration of water occurs if there is an unequal distribution of
at least one dissolved substance on either side of a membrane and the membrane is
impermeable to that substance. In this situation, the substance is called an osmotically
active substance (OAS). For example, if a membrane that is impermeable to sucrose
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separates a solution of sucrose from distilled water, water will move from the distilled water,
where it is in higher concentration through the membrane into the solution, where it is in
lower concentration (due to the presence of the sucrose molecules). In this case, sucrose is
the osmotically active substance.
Three terms, hypertonic, hypotonic, and isotonic, are used when referring to two
solutions separated by a selectively permeable membrane (Figure 3.1). The hypertonic
solution (Figure 3.1a) has a greater concentration of OAS than the solution on the other side
of the membrane. It is described, therefore, as having a greater osmolarity (solute
concentration expressed as molarity). The hypotonic solution (Figure 3.1b) has a lower
concentration of OAS, or a lower osmolarity, than the solution on the other side of the
membrane. When the two solutions are in equilibrium, the concentration of OAS being equal
on both sides of the membrane, the osmolarities are equal and the substances are said to be
isotonic (Figure 3.1c). The net flow of water is from the hypotonic to the hypertonic solution.
When the solutions are isotonic, there is no net flow of water across the membrane.
The concept of osmotic pressure must be understood when studying osmosis.
The movement of water from a hypotonic solution through the membrane into a hypertonic
solution can be prevented by applying force or pressure on the hypertonic side (Figure 3.2).
The force that must be applied to prevent osmotic movement of water from a hypotonic to
hypertonic solution, measured in atmospheres, is referred to as osmotic pressure. Solutions
with greater concentrations of OAS have greater osmotic pressures because greater force is
required to prevent water movement into them. Distilled water has an osmotic pressure of
zero.
Figure 3.1 Diagrammatic representation of cells in (a) hypertonic, (b) hypotonic, and (c)
isotonic solutions.
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Figure 3.2 Applying the correct pressure to the hypertonic side of two solutions separated by
a semi-permeable membrane will prevent water movement into the hypertonic solution.
Experiment 3A
Diffusion of Molecules Through a Selectively Permeable Membrane
Materials
30 cm strip of moist dialysis

tubing Starch solution
I2Kl solution
String or rubber band
Glucose Test Strips
Wax pencil
Three standard test tubes
10% glucose solution
Disposable transfer pipettes
500 ml beaker, one-third filled
Two 400 ml beakers to hold dialysis
with water
bag
Hand-held test tube holder
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Introduction
Dialysis tubing is a membrane made of regenerated cellulose fibers

formed into a flat tube. If two solutions containing dissolved substances of
different molecular weights are separated by this membrane, some substances
may readily pass through the pores of the membrane, but others may be
excluded.
Working in teams of four students, you will investigate the selective
permeability of dialysis tubing. You will test the permeability of the tubing to
glucose (molecular weight 180), starch (a variable-length polymer of glucose),
and iodine potassium iodide (I2KI). You will place a solution of glucose and
starch into a bag made from dialysis tubing, and then place this bag into a
solution of I2KI. Sketch and label the design of this experiment in the margin of
your lab manual to help you develop your hypotheses.
You will use two tests in your experiment:

1. I2KI test for presence of starch. When I2KI is added to the unknown
solution, the solution turns purple or black if starch is present. If no
starch is present, the solution remains a pale yellow-amber color.
2. Glucose strip monitor for presence of glucose. Glucose
concentrations can be monitored using a paper-based sensing
device.
Hypothesis
With your group, hypothesize about the selective permeability of dialysis
tubing to the substances being tested. Keep a record of your hypotheses.
Prediction
With your group, predict the results of the I2KI and glucose strip tests
based on your hypotheses (if/then). Keep a record of your predictions.
Procedure
1. Prepare the dialysis bag with the initial solutions:
a. Fold over 3 cm at the end of a 25 to 30 cm piece of dialysis tubing
that has been soaking in water for a few minutes, pleat the folded
end “accordion style,” and close the end of the tube with the string
or a rubber band, forming a bag. This procedure must secure the
end of the bag so that no solution can seep through (Figure 3.3).
b. Roll the opposite end of the bag between your fingers until it
opens, and add 4 pipettes full of 10% glucose into the bag. Then
add 4 pipettes full of starch solution to the glucose in the bag.
c. Record the contents of the bag and the beaker in Table 3.1. Your
lab instructor will prepare a control bag containing only distilled
water; record its contents in Table 3.1 as well.
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d. Hold the bag closed and mix its contents. Record its color in
Table 3.1 in the Results section. Carefully rinse the outside of the
bag in tap water.
e. Add 300 ml of water to a 500 ml beaker. Add several droppers full
of I2KI solution to the water until it is visibly amber. Record the
color of the H20
+I2KI solution in Table 3.1.
f. Place the bag in the beaker so that the untied end of the bag hangs
over the edge of the beaker (Figure 3.3). Do not allow the liquid to
spill out of the bag! If the bag is too full, remove some of the liquid
and rinse the outside of the bag again. If needed, place a rubber
band around the beaker, holding the bag securely in place. If some
of the liquid spills into the beaker, dispose of the beaker water,
rinse, and fill again.
2. Leave the bag in the beaker for about 30 minutes (you should go to
another lab activity and then return to check your setup periodically).
3. After 30 minutes, carefully remove the bag and stand it in a dry beaker.
4. In Table 3.1, record the final color of the solution in the bag and the final
color of the solution in the beaker. Observe the control bag sitting in H20
+I2KI solution, and record the color of its contents as well.
5. Perform the glucose strip test for the presence of sugar in the solutions:
a. Label three glucose strips: control, bag and beaker.
b. Carefully dip the thick end of the appropriate strip into the liquid from
each sample. Keep the end submerged for only 1-2 seconds.
c. Allow the strips to dry for 30 seconds – 1 minute.
d. Record the color of the glucose test strips in Table 3.1
Next, review your results in Table 3.1 and draw your conclusions
in the Discussion section.
Figure 3.3. Experimental setup for Experiment 3A.
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Experiment 3B. Osmotic Behavior in Cells
Materials:
2 compound microscopes labeled A and B

1 slide of Elodea in salt solution
1 slide of Elodea in distilled water
Introduction
In their natural environment, cells of freshwater plants and algae are bathed in
water with a low concentration of OAS. The net flow of water is from the
surrounding medium into the cells.
The persistence of a cell wall and a large fluid-filled central vacuole in a plant or
algal cell will affect the cell’s response to solutions of differing molarities. When a
plant cell is placed in a hypertonic solution, water moves out of the cell; the
protoplast shrinks and may pull away from the cell wall. This process is called
plasmolysis, and the cell is described as plasmolyzed. In a hypotonic solution, as
water moves into the cell and ultimately into the cell’s natural vacuole, the cell’s
protoplast (the plant cell exclusive of the cell wall – the cytoplasm enclosed by
plasma membrane) expands. The cell wall, however, restricts the expansion,
resulting in turgor pressure (pressure of the protoplast on the cell wall owing to
uptake of water). A high turgor pressure will prevent further movement of water
into the cell. This process is a good example of the interaction between pressure
and osmolarity in determining the direction of the net movement of water. The
hypertonic condition in the cell draws water into the cell until the membrane-
enclosed cytoplasm presses against the cell wall. Turgor pressure begins to
force water through the membrane and out of the cell, changing the direction of
the net flow of water (Figure 3.4).
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Figure 3.4. Changes in a hypertonic plant cell placed in hypotonic solution: (a)
net flow of water is into plant cell (thick arrows), and membrane-enclosed
cytoplasm begins to expand towards cell wall (thin arrows); (b) water continues
to move into the hypertonic cell, filling the vacuole and further pushing the
membrane-bound cytoplasm against the cell wall; (c) the filled vacuole pushes
the cytoplasm and plasma membrane against the rigid cell wall, exerting turgor
pressure that forces water out of the cell. This turgor pressure is equivalent to
the osmotic pressure of the cell, preventing further expansion (equal arrows).
For this experiment, two slides have been set up on demonstration

microscopes. On each slide, Elodea has been placed in a different molar
solution: One is hypotonic (distilled water) and one is hypertonic (concentrated
salt solution).
Hypothesis
With your group, hypothesize about the movement of water in cells with a cell
wall when they are placed in hypertonic or hypotonic environments. Record your
hypotheses.
Prediction
With your group, predict the appearance of Elodea cells placed in the two
solutions (if/then). Record your predictions.
Procedure
1. Observe the two demonstration microscopes with Elodea in solutions A
and B.
2. Record your observations in the results section, and interpret your
results in the Discussion section.
23
Experiment 3C. Estimating the Osmolarity of Plant Cells
Frequently, plant scientists need to determine the optimum water content for
normal physiological processes in plants. They know that for normal activities
to take place, the amount of water relative to osmotically active substances in
cells must be maintained within a reasonable range. If plant cells have a
reduced water content, all vital functions slow down.
In the following experiment, you will estimate the osmolarity of potato tuber cells
via their change in water weight. You will incubate pieces of potato tuber in
sucrose solutions of known molarity. The object is to find the molarity at which
weight or volume of the potato tuber tissue does not change, indicating that
there has been no net loss or gain of water. This molarity is an indirect measure
of the osmolartiy of the potato tuber.
Work in teams of four. Time will be available near the end of the laboratory
period for each team to present its results to the class for discussion and
conclusions.
Materials
One large potato tuber Sucrose solutions: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 molar (M)
250 ml beakers or cups Razor blade
Wax marking pencil Cork borer
Forceps Deionized (DI) water
Paper towels Balance (nearest 0.01 g)
Metric ruler Petri dish
Aluminum foil Calculator
Introduction
In this experiment, you will determine the weight of several potato tuber
cylinders and incubate them in a series of sucrose solutions. After the cylinders
have incubated, you will weigh them and determine if they have gained or lost
weight. This information will enable you to estimate the osmolarity of the potato
tuber tissue.
Procedure
1. Obtain 100 ml of DI water and 100 ml of each of the sucrose solutions.
Put each solution in a separate, appropriately labeled 250 ml beaker or
paper cup.
Note: Cork borers and razor blades can cut! Use them with extreme care!
To use the cork borer, hold the potato in such a way that the borer will not
push through the potato into your hand.
2. Use a sharp cork borer to obtain seven cylinders of potato. Push the borer
through the length of the potato and push the potato cylinder out of the
24
borer. You must have seven complete, undamaged cylinders at least 5
cm long.
3. Line up the potato cylinders and, using a sharp razor blade, cut all
cylinders to a uniform length, about 5 cm, removing the peel from the
ends.
4. Place all seven potato samples in a Petri dish, and keep them covered
to prevent their drying out.
Note: in subsequent steps, treat each sample individually. Work quickly.
To provide consistency, each person should do one task to all cylinders
(one person wipe, another weigh, another slice, another record the
data).
5. Remove the cylinder from the Petri dish, and place it between the folds of
a paper towel to blot sides and ends.
6. Weigh it to the nearest 0.01 g on the aluminum sheet on the balance.
Record the weight in Table 3.2 in the Results section.
7. Immediately cut the cylinder lengthwise into two long halves.
8. Transfer potato pieces to the water beaker.
9. Note what time the potato pieces are placed in the water beaker.
10. Repeat steps 5 to 8 with each cylinder, placing potato pieces in the
appropriate incubating sucrose solution from 0.1 to 0.6 M.
Note: Be sure that the initial weight of the cylinder placed in each test
solution is accurately recorded.
11. Incubate 1.5 to 2 hours (As this takes place, you will be performing
other lab activities).
12. Swirl each beaker every 10 to 15 minutes as the potato pieces incubate.
13. At the end of the incubation period, record the time when the potato
pieces are removed.
14. Remove the potato pieces from the first sample. Blot the pieces on a
paper towel, removing excess solution only.
15. Weigh the potato pieces and record the final weight in Table 3.2.
16. Repeat this procedure until all samples have been placed in the test
solutions.
17. Analyze your data in Table 3.2, and complete the questions in the
Discussion section.
25
Exercise 3A Worksheet
Complete Table 3.1 as you observe the results of this experiment.
Fill in a title for Table 3.1.
Solution Contents Original Final Color Glucose

Source of Source Color Strip
Color
Bag
Beaker
Control Bag
Table 3.1.
Discussion
1. What is the significance of the final colors and the colors after the
glucose strip tests? Did the results support your hypothesis? Explain,
giving evidence from the results of your tests.
2. How can you explain your results?
3. From your results, predict the size of I2KI molecules relative to

glucose and starch.
4. What colors would you expect if the experiment started with glucose
and I2KI inside the bag and starch in the beaker? Explain.
26
Exercise 3B Worksheet
Draw the appearance of Elodea cells in solutions A and B. Record any
additional observations.
Solution A Solution B
Discussion
1. Based on your predictions and observations, which solution is hypertonic?
Which solution is hypotonic?
2. Which solution has the greatest osmolarity?
3. Would you expect pond water to be isotonic, hypertonic or hypotonic to

Elodea cells? Explain.
4. Verify your conclusions with your laboratory instructor.
27
Exercise 3C Worksheet
1. Complete Table 3.2 and provide a title. To calculate percentage change in

weight, use this formula:
Weight change = final weight – initial weight
Percentage change in weight = (weight change/initial weight) X 100
If the sample increased in weight, the value should be positive. If it

decreased in weight, the value should be negative.
Table 3.2
2. Next week, you will plot percentage change in weight as a function of the
sucrose molarity using Microsoft Excel. These results will be used during the
quantitative methods workshop.
Discussion (to be completed next week)
1. At what sucrose molarity does the curve cross the zero change line on
the graph?
2. Explain how this information can be used to determine the osmolarity of

the potato tuber tissue.
3. Estimate the osmolarity of the potato tuber tissue.
28
Exercise 4:
Yeast Respiration and Fermentation
1. Understand the process of alcoholic fermentation in yeast, and under

what conditions these organisms switch from aerobic respiration to
fermentation.
2. Estimate the rate of fermentation as it relates to the rate of carbon
dioxide production, and to understand/demonstrate the relationship
between yeast concentration and fermentation rate.
Alcohol Fermentation in Yeast
For centuries, humans have taken advantage of yeast fermentation to produce

alcoholic beverages and bread. Consider the products of fermentation and their
roles in making these economically and culturally important foods and beverages.
Alcoholic fermentation begins with glycolysis, a series of reactions breaking
glucose into two molecules of pyruvate with a net yield of 2 ATP and 2NADH
molecules. In anaerobic environments, in two steps the pyruvate (a 3-carbon
molecule) is converted to ethyl alcohol (ethanol, and
2-carbon molecule) and CO2. In this process the 2 NADH molecules are
oxidized, replenishing the NAD+ used in glycolysis.
Materials
4 respirometers: test tubes, 1 ml graduated pipettes, aquarium tubing, flasks,

binder clips pipette pump
3 – 5ml graduated pipettes, labeled “DI water”, “yeast” and “glucose”
3 inch donut-shaped metal weights
yeast solution
glucose
solution DI
water
water bath
wax pencil
29
Introduction
In this lab study, you will investigate alcoholic fermentation in a yeast (a single-
celled fungus), Saccharomyces cerevisiae, or “baker’s yeast”. When oxygen is
low, some fungi (including yeast) and most plants, switch from cellular respiration
to alcoholic fermentation. In bread making, starch in the flour is converted to
glucose and fructose, which then serve as the starting compounds for
fermentation. The resulting carbon dioxide (CO2) is trapped in the dough,
causing it to rise. Ethanol is also produced in bread making but evaporates
during baking.
In this laboratory experiment, the CO2 gas produced bubbles out of the solution
and can be used as an indication of the relative rate of fermentation taking place.
Figure 9.1 illustrates the respirometers you will construct and use to collect CO 2.
The rate of fermentation, a series of enzymatic reactions, can be affected by
several factors, for example concentration of yeast, concentration of glucose, or
temperature. In this lab study, you will investigate the effects of yeast
concentration. In your independent study you may choose to investigate other
independent variables.
Figure 4.1
Hypothesis
With your group, hypothesize about the effect of different concentrations of
yeast on the rate of fermentation. Record your hypothesis.
30
Prediction
With your group, predict the results of the experiment based on your hypothesis
(if/then). Record your predictions.
Procedure
1. Obtain four flasks and add enough tap water to keep them from floating in
a water bath (fill to about 5 cm from the top of the flask). Label the flasks
1, 2, 3, and 4. To stabilize the flasks, place a 3-inch donut-shaped metal
weight over the neck of the flasks.
2. Obtain four test tubes (fermentation tubes) and label them 1, 2, 3, and
4. Add solutions as in Table 4.1 to the appropriate tubes. Rotate each
tube to distribute the yeast evenly in the tube. Place tubes in the
corresponding numbered flasks.
3. To each tube, add a 1 ml graduated pipette to which a piece of plastic
aquarium tubing has been attached.
4. Place the flasks with the test tubes and graduated pipettes in the
water bath at 30◦C. Allow them to equilibrate for about 5 minutes.
Table 4.1 Contents of fermentation solutions (volumes in ml)
5. Attach the pipette pump to the free end of the tubing on the first pipette.
Use the pipette pump to draw the fermentation solution up into the
pipette. Fill it past the calibrated portion of the tube, but do not draw the
solution into the tubing. Fold the tubing over and clamp it shut with the
binder clip so the solution does not run out. Open the clip slightly, and
allow the solution to drain down to the 0 ml calibration line (or slightly
below). Quickly do the same for the other three pipettes. [Note: if you
cannot stabilize the set up at the 0 ml line, keep track of where you
started (this will be your initial reading)].
6. In Table 4.2, quickly record your initial readings for each pipette in the
“initial reading” row in each “Actual (A)” column. This is the initial time (I).
7. Two minutes after the initial readings for each pipette, record the actual
readings
(A) in ml for each pipette in the “Actual (A)” column. Subtract I from A to
determine the total amount of CO2 evolved (A-I). Record this value in
the “CO2 Evolved (A-I)” column. From now on, you will subtract the
initial reading from each actual reading to determine the total amount of
31
CO2 evolved.
8. Continue taking readings every 2 minutes for each of the solutions for 20
minutes. Remember, take the actual reading from the pipette and subtract
the initial reading
(I) to get the total amount of CO2 evolved in each test tube.
9. Record your results in Table 4.2
#10 and #11 Below will be completed during Recitation next week.
10. Construct a graph in Figure 4.2

11. Answer discussion questions on page 33-35.
32
Exercise 4 Worksheet
1. Complete Table 4.2 and provide a title. [Carefully read instructions in
the procedure. To figure out the amount of CO2 evolved, make sure to
subtract the same initial reading (I) from the Actual reading (A) at each
time point].
Table 4.2
2. Construct a graph to illustrate your results (Label Figure 4.2). [plot

results from all 4 tubes on a single graph]
a. What is (are) the independent variable(s)? Which is the
appropriate axis for the variable?
b. What is the dependent variable? Which is the appropriate axis
for this variable?
c. Choose an appropriate scale and label the x (horizontal) and y
(vertical) axes.
d. Should you use a legend? If so, what would this include?
e. Compose a figure title.
33
Figure 4.2
34
Discussion
1. Explain the experimental design. What is the purpose of each

test tube? Which is (are) the control tube(s)?
2. Which test tube had the highest amount of fermentation (largest

increase in CO2)? Explain why.
3. Which test tube had the lowest amount of fermentation (smallest

increase in CO2)? Explain why.
4. Why are the different amounts of water added to each fermentation

solution?
35
Exercise 5:
Bacterial Growth Analysis

1. Research and identify information on genes
2. Be able to generate a hypothesis and revise the hypothesis based on results
3. Calculate how much of culture to add to normalize cell density
4. Determine optical density of bacterial cultures
5. Perform microscopic analysis of bacterial cultures
6. Graph and interpret data
7. Gain presenting experience
Introduction
Cyanobacteria are a group of photosynthetic bacteria and the first organisms on the planet
to be able to perform oxygenic photosynthesis, resulting in the oxygen rich environment we
live in today. Moreover, plants acquired the ability to perform oxygenic photosynthesis
through an endosymbiotic relationship with a cyanobacterium resulting in the chloroplast.
Currently, cyanobacteria produce ~30% of the atmospheric oxygen we breathe! In this lab
exercise we will be monitoring the growth of one particular cyanobacterial species,
Synechococcus elongatus PCC 7942, or S. elongatus for short. S. elongatus is used by
many academic and industrial laboratories to study a variety of biological processes,
including photosynthesis, biofuel production, the circadian clock and cell division, because
it is easy to grow in the lab, has a fully sequenced genome and can be easily genetically
modified (Figure 1).
Figure 1. S.
elongatus growing
on agar plates
(left) or in liquid
culture (right)
In this exercise you will set up an experiment where you and your group will compare the
growth and cell shape of two mutant S. elongatus strains to wild-type.
36
Materials
Part A:
Computer with internet access
Spectrophotometer set to read OD750
Bacterial cultures (inoculated 1-week before)
 TA will measure OD750 of cultures prior to class and report numbers
to the class
16x100mm test tubes with loose fitting caps filled with 3mL BG-11 media (3 per
group)
16x100mm test tubes with loose fitting caps filled with 3mL BG-11 media + 5ug/mL
Kanamycin (6 per group)
Micropipette tips
Gloves
Part B:
Computer with excel installed
Cuvettes (10 per group)
BG-11 media
Cultures from Part 1
Microscope slides
Cover slips
Immersion oil
Microscope with various objectives including 60X and 100X
Micropipette tips
Gloves
Procedure
Exercise 5: BACTERIAL GROWTH ANALYSIS - PART A
Identifying mutants of interest

Your group is tasked with:
1. Reviewing the list of mutant strains available to choose from (Table 1)
2. Research the genes, by reading the text book or other reputable sources of
information (journal articles etc.). Note you may not be able to read an article about
your gene of interest in S. elongatus but you may find an article that studies that
gene in another organism!
3. Synthesize the research that you have done and choose two mutant strains that
you are interested in investigating for their growth and cell shape properties.
4. Formulate a hypothesis for how you think your selected strains will grow and look,
relative to each other as well as a wild-type (WT) control, for a total of 3 cultures.
Be sure to include a rationale to support your hypothesis.
5. Present your experimental setup, which mutant strains you chose and why you
chose them, as well as your hypothesis to the class.
37
Strain Description of strain Function of deleted gene
WT Wild-type S. elongatus strain N/A
clpX::Km clpX gene has been deleted from the Protein
chromosome and replaced with chaperone/degradation
Kanamycin (Km) resistance cassette
kaiA::Km kaiA gene has been deleted from the Circadian clock
chromosome and replaced with
smtA::Km smtA gene has been deleted from the Metallothionein- Zn/Cu
chromosome and replaced with homostasis/ oxidative stress
Kanamycin (Km) resistance cassette protection
mreD::Km mreD gene has been deleted from the Bacterial actin homolog
hlyD::Km hylD gene has been deleted from the Protein secretion
smc::Km smc gene has been deleted from the Structural maintenance of
chromosome and replaced with chromosomes
sbbC::Km sbbC gene has been deleted from the exonuclease
rbp2::Km rbp2 gene has been deleted from the RNA binding protein 2
Table 1. List of available strains to choose from
Inoculating cultures
After your group has selected the two mutant strains you want to compare the next step is
to inoculate your strains as well as a wild-type control. You will need to inoculate each strain
in triplicate, meaning that you will set up 3 cultures for each strain, for a total of 9 cultures.
Biological replicates, in this case triplicate cultures, are important for measuring the variation
of your experiment so that statistical analysis can be applied to evaluate differences.
1. Label 9 test tubes with your strain and well as replicate number (1, 2 or 3). Be sure
to include lab section and group identifier.
a. The 3 tubes that will contain the wild-type strain should contain BG-11
media without antibiotics
b. The 6 tubes that will contain your mutant strains should contain BG-11
supplemented with the antibiotic Kanamycin (Km), which has replaced your
gene of interest, resulting in the deletion of your gene of interest
2. Calculate how much of the starting culture you will need to add for each of your
cultures to obtain a starting optical density of 0.05
a. You will be given the optical density reading at 750nm (OD750), which is a
proxy for cell density, of the cultures you will use to inoculate your
experimental cultures
38
b. You want your experimental cultures to each have a starting density (OD750)
of 0.05 in a total volume of 3mL
c. Hint- You will have to use the equation C1V1=C2V2
3. Based on your calculations add the appropriate amount of the culture to each of
your experimental tubes and give to your instructor. They will be grown at 30℃ in
constant light conditions. Remember that the bacteria are photosynthetic, meaning
they require light to grow!
Lab instructors: Collect strains and give to IST to grow the cultures in Rosser Hall
357.
39
Exercise 6: BACTERIAL GROWTH ANALYSIS - PART B
Materials
Part B:
Computer with excel installed
Cuvettes (10 per group)
BG-11 media
Cultures from Part 1
Microscope slides
Cover slips
Immersion oil
Microscope with various objectives including 60X and 100X
Micropipette tips
Gloves
Measuring optical density of your cultures

1. Collect 10 cuvettes-one for each of your samples as well as one that will contain
your “blank”
a. To one cuvette add 1mL of BG-11 media. This will be your “blank” sample
which will be used to calibrate the spectrophotometer and set the solution’s
absorbance as zero. Therefore, any number greater than zero can be
determined to be bacterial growth.
b. For each of your 9 cultures add 1mL to a clean cuvette
2. Measure the OD750 of each of your samples, be sure to write down the numbers in
your lab notebook!
Plot Data in EXCEL
1. Enter your data into an excel spread sheet. An example of how to set up your
spread sheet is shown in Figure 2.
a. Remember that all your cultures initially started at an OD750 of 0.05
Figure 2. Example of how to set up your excel spread

sheet and plotting of data
2. Calculate the average OD750 for the three replicates for each of your samples. An
example is shown in Figure 3.
a. Create a line to enter the average
b. Type in =average(highlight the cells you want to average)
c. Hit enter
40
Figure 3. Example of how to calculate the average of
your three replicates.
3. Calculate the standard deviation of your samples. An example is shown in Figure 4.

a. Create a line to enter standard deviation
b. Type in=stdev(highlight the cells you want to calculate standard deviation)
c. Hit enter
Figure 4. Example of how to calculate the standard

deviation of your three replicates.
4. Plot your data. An example is shown in Figure 5.

a. Copy and paste your average OD750 numbers to create a new chart listing
just the average number of your average OD750 from your triplicate cultures.
b. Highlight your data and in the “Insert” tab at the top select 2D line with
markers chart from the Charts options
c. In the “Design” tab at the top select Switch Row/Column in the Data options
Figure 5. Example of how plot your data.
41
5. Add error bars to your graph. An example is shown in Figure 6.
a. With the graph selected, look in the “Design” tab at the top
i. Under “Add Chart Element”, select “Error Bars”
ii. Select “More Error Bar Options”, this will open a window to select
the series
iii. Select the strain you want to enter error bars for. Start with the wild
type strain then repeat for each of the mutants
1. Select WT then click OK
2. This will open a “Format Error Bars” panel
iv. Under “Error Bar Options” you will select the “Vertical Error Bar” tab,
this tab should be open by default but you can select the three bars
on top if it does not
v. The default settings should be “Both” for “Direction” and “Cap” for
“End Style”. These are both good settings to use.
vi. For “Error Amount” select custom and click on the “Specify Value”
box, this will open a new window
vii. For both the “Positive Error Value” and “Negative Error Value”
highlight the cells that have your calculated standard deviation for
the series you are working with (either WT, mut-1 or mut-2)
1. You may need to clear the default entry before highlighting
the cells of interest
2. Click OK
3. You should see error bars on your graph
Figure 6. Example of how to plot data and error bars on

graph.
Image strains under the microscope
1. Start by looking at the color of your cultures.

a. Do they all look the same shade of green?
42
i. The green color comes from the photosynthetic pigments. Can you
make any conclusions about the photosynthetic activity of these
cultures by looking at them?
2. In order to image your strains on the microscope, collect glass slides, cover slips
and light microscope.
3. For each strain, you may only want to check one or two of your replicates, add 5uL
of liquid culture to the middle of a clean glass microscope slide
4. Cover your drop of cells with a cover slip
5. Use microscope to focus on cells and observe cell shape
a. S. elongatus is typically ~2-5um long, thus you will see it best on the 100x
objective. However, it might be easier to identify cells on a lower
magnification (10x or 40x) objective first.
6. Record your observations by drawing in your lab notebook and taking pictures with
your cell phone
Present your results to the class
Take the time to analyze your data and prepare a small presentation that addresses the
following questions:
1. What was your original hypothesis?

2. Describe your results?
a. How did your mutant strains grow? Compare growth of the WT strain to
each mutant and each mutant to each other. Which grew best and which
grew worst?
b. What cell morphology did your mutant strains have compared to the
wild-type strain?
c. How did cell morphology/shape correlate with growth?
3. What can you conclude about the function of the genes that was deleted in your
mutant strains?
4. How do your results compare with your original hypothesis?
5. Revise your hypothesis and suggest an experiment to test your hypothesis
Present your findings to the class. Both you and the instructor should keep a record of how
the different mutants strains tested by the class grew and what the cell morphology was.
As a class can you come up with any conclusions? Can you correlate how a strain will
grow based on its morphology?
43
Exercise 7:
Using a Single-Nucleotide Polymorphism to
Predict Bitter-Tasting Ability
Preparation for This Laboratory Unit
This laboratory topic spans 3 weeks:

Week 1 – Preparation of cheek cell DNA for polymerase chain reaction (PCR)
Week 2 – Set up of PCR reaction
Week 3 – Restriction enzyme digestion and gel electrophoretic analysis of PCR reaction
Before coming to Week 1 of lab, be sure to read over this entire section completely.
General Introduction
Mammals are believed to distinguish only five basic tastes: sweet, sour, bitter, salty,
and umami (the taste of monosodium glutamate). Taste recognition is mediated by
specialized taste cells that communicate with several brain regions through direct
connections to sensory neurons.
Taste perception is a two-step process. First, a taste molecule binds to a specific
receptor on the surface of a taste cell. Then, the taste cell generates a nervous
impulse, which is interpreted by the brain. For example, stimulation of “sweet cells”
generates a perception of sweetness in the brain. Recent research has shown that
taste sensation ultimately is determined by the wiring of a taste cell to the cortex, rather
than the type of molecule bound by a receptor. So, for example, if a bitter taste receptor
is expressed on the surface of a “sweet cell,” a bitter molecule is perceived as tasting
sweet.
A serendipitous observation at DuPont, in the early 1930s, first showed a genetic basis
to taste. Arthur Fox had synthesized some phenylthiocarbamide (PTC), and some of
the PTC dust escaped into the air as he was transferring it into a bottle. Lab-mate C.R.
Noller complained that the dust had a bitter taste, but Fox tasted nothing—even when
he directly sampled the crystals. Subsequent studies by Albert Blakeslee, at the
Carnegie Department of Genetics (the forerunner of Cold Spring Harbor Laboratory),
showed that the inability to taste PTC is a recessive trait that varies in the human
population.
Bitter-tasting compounds are recognized by receptor proteins on the surface of taste

cells. There are approximately 30 genes for different bitter taste receptors in
mammals. The gene for the PTC taste receptor (which is a protein), TAS2R38, was
identified in 2003. Sequencing identified three nucleotide positions that vary within the
human population—each variable position is termed a single nucleotide
polymorphism (SNP). One specific combination of the three SNPs, termed a
haplotype, correlates most strongly with tasting ability.
The principle in this context is that single nucleotide differences in a human gene
sequence give rise to single amino acid differences in human protein sequences,
which give rise to different abilities to taste bitter compounds. Although the DNA from
44
different humans is more alike than say the DNA of a human and a mouse, there are
clearly many variations in the DNA of different individual humans. These variations
contribute to our diversity (such as our ability to taste bitter compounds). Such
variations are termed "polymorphisms" (meaning many forms). Here in this lab, we
are interacting with single nucleotide polymorphisms (SNPs) as described in the
preceding paragraph. Some of these variations provide the basis for studies of
population genetics, genetic disease diagnosis, forensic identification, and paternity
testing. In this lab, the variation we are studying is the basis for differing abilities to
taste bitter compounds. Analogous changes in other cell-surface molecules influence
the activity of many drugs. For example, SNPs in serotonin transporter and receptor
genes predict adverse responses to anti-depression drugs, including PROZAC® and
Paxil®.
In this experiment, we will first obtain a sample of human cells by using a saline
mouthwash. DNA is extracted from this sample of cells by boiling it with Chelex resin.
This resin binds contaminating metal ions. At the end of this process, each student will
have a sample of their DNA in a plastic tube.
Then, we will perform the polymerase chain reaction (PCR) to amplify a short region of
the TAS2R38 gene. At the end of this process, each student will have a large amount
of only this short region of the TAS2R38 gene.
Next, the amplified PCR product (the short region of the TAS2R38 gene) will be cut
(cutting is called “digestion”) with the restriction enzyme HaeIII, whose recognition
sequence includes one of the SNPs. This enzyme identifies a short sequence within
the TAS2R38 gene and physically cuts the double stranded DNA at that site. However,
the SNP is located at this very site. Therefore, the enzyme can only recognize one
allele (SNP) at this location and not the other allele (SNP) at this location. People who
possess the ability to taste bitter compounds using the taste receptor encoded by this
gene (called “tasters”) possess the SNP that is cut by the HaeIII enzyme. People who
do not possess the ability to taste bitter compounds using the taste receptor encoded
by this gene (called “nontasters”) possess the SNP that is not cut by the HaeIII
enzyme). Thus, one allele is cut by the enzyme (“taster” SNPs), and one is not
(“nontaster” SNPs).
After the enzyme digestion (called a restriction digestion), the products of the enzyme
digestion will be separated using an agarose gel. Because the taster SNP is cut by the
enzyme and the nontaster SNPs are not cut by the enzyme, students will be able to
identify their genotypes (taster SNPs or nontaster SNPs) by looking at whether they
have one (nontaster) or multiple (taster) pieces of DNA on the agarose gel. This is
called a restriction fragment length polymorphism (RFLP).
Each student scores his or her genotype, predicts their tasting ability, and then tastes
PTC paper. Students with nontaster SNPs should not sense a bitter taste when tasting
this paper, while students with taster SNPs will be able to sense a bitter taste. Class
results show how well PTC tasting actually conforms to classical Mendelian
inheritance, and illustrates the modern concept of pharmacogenetics—where a SNP
genotype is used to predict drug response.
45
Exercise 7 (Part A): Isolation of nuclear DNA from your own cheek cells

1. State the purpose of the DNA isolation
2. Explain the purpose(s) of the heat, centrifugation, and Chelex steps in
the procedure.
Introduction
First and foremost, it is necessary to have a sample of DNA containing the segment you
wish to amplify. This DNA is called the template because it provides the pattern of base
sequence to be duplicated during the PCR process. The source of DNA is a sample of
several thousand cells from your cheek, obtained by saline mouthwash. The cells are
collected by centrifugation and resuspended in a solution containing the resin "Chelex,"
which binds metal ions that inhibit the PCR reaction. The cells are lysed by boiling and
centrifuged to remove cell debris. A sample of the supernatant containing DNA from the
nucleus is mixed with the other reagents for the PCR reaction.
Materials
Microcentrifuge for 1.5 ml tubes with adaptors for PCR tubes Thermocycler set at
◦
99 C
Thermocycler position grid sheet
Vortexers
Labeled storage box for storage of collected, labeled, individual DNA samples.
For each student:

200 microliters (µL) 5% Chelex in a PCR tube
0.9% saline – 10 ml in a STERILE paper cup [this should be setup in hallway, no
drinking in lab]
1.5 mL microfuge tubes (2)
For each group of 4 students:

150 µL 0.9% saline in a microfuge tube
Permanent marker
46
Procedures: DNA Isolation
47
48
At this point, have your instructor place the labeled tube containing your DNA
sample in the box for storage until the next class meeting.
49
Exercise 8 (Part B): PCR – Polymerase Chain Reaction
Advance Preparation
Before coming to Week 2 of this laboratory exercise, be sure to reread this section of
the chapter. Also, be sure to observe the animation of PCR at
https://www.youtube.com/watch?v=DkT6XHWne6E and answer the questions in this
section of the manual. You are required to turn in these completed questions at the
beginning of lab.
1. state the purpose of PCR

2. list and explain the function of each component in the PCR reaction
3. name and describe what is happening at each of the different
temperatures in a PCR cycle
4. state what has happened to the quantity of DNA at the end of each cycle
Introduction
The polymerase chain reaction (PCR) is a method used by scientists to rapidly copy, in
a test tube, specific segments of DNA. By mimicking some of the DNA replication
strategies employed by living cells, PCR has the capacity for churning out millions of
copies of a particular DNA region. It has found use in population genetics, forensic
science, in the diagnosis of genetic disease, and in the cloning of rare genes. One of the
reasons PCR has become such a popular technique is that it doesn’t require much
starting material. It can be used to amplify DNA recovered from a plucked hair, from a
small spot of blood, or from the back of a licked postage stamp.
There are some essential reaction components and conditions needed to amplify DNA
by PCR. Along with template DNA, PCR requires two short single-stranded pieces of
DNA called primers. These are usually about 20 bases in length and are
complementary to opposite strands of the template at the ends of the target DNA
segment being amplified.
Primers attach (anneal) to their complementary sites on the template and are used as
initiation sites for synthesis of new DNA strands. Notice that these primers are different
from each other and are chosen to bind to opposite strands of the DNA.
Deoxynucleotide triphosphates containing the bases A, C, G, and T (dATP, dCTP,
dGTP, and dTTP) are also added to the reaction. The enzyme Taq DNA polymerase
binds to one end of each annealed primer and strings the deoxynucleotides together to
form new DNA chains complementary to the template. Only one end of the primer has
the appropriate chemical structure for adding on new nucleotides. The DNA
polymerase enzyme absolutely requires the metal ion magnesium (Mg++) for its
activity. It is supplied to the reaction in the form of MgCl2 salt. A buffer is used to
50
maintain an optimal pH level.
PCR is accomplished by repeatedly cycling a reaction through three temperature

steps. In the first step, denaturation, the two strands of the template DNA molecule are
separated, or denatured, by exposure to a high temperature (usually 94° to 96°C). Once
in a single-stranded form, the bases of the template DNA are exposed and are free to
interact with the primers. In the second step of PCR, called annealing, the reaction is
brought down to a temperature usually between 37°C to 55 °C. At this lower
temperature, stable hydrogen bonds can form between the complementary bases of the
primers and template. Although human genomic DNA is billions of base pairs in length,
the primers require only seconds to locate and anneal to their unique complementary
sites. In the third step of PCR, called extension, the reaction temperature is raised to an
intermediate level (65°C to 72°C), at which the Taq DNA polymerase enzyme can
efficiently extend the length of the DNA primer by addition of nucleotides. The DNA
polymerase adds nucleotides, one at a time, to the accepting end of each primer. These
three phases are repeated over and over again, doubling the number of DNA molecules
with each cycle. After 25 to 40 cycles, millions of copies of DNA are produced. The PCR
process taken through four cycles is illustrated on the following page (Figure 1).
In this procedure, you will amplify either a 400-bp region and/or a 100-bp region from
within a gene on Chromosome 8. Which fragment sizes are amplified in your reaction
depends upon which allele(s) are present in your DNA. Each of the three possible
genotypes—homozygous for the short fragment, homozygous for the long fragment,
or heterozygous (one short and one long fragment) can be identified following
electrophoresis in an agarose gel, which we will do next week.
51
52
Student Name
Pre-Lab PCR Homework (page 1 of 2)
You should be able to answer the questions below after reading the introductory
material and careful viewing of the PCR tutorial at
https://www.youtube.com/watch?v=DkT6XHWne6E
1. What is the purpose of PCR?
2. How are the strands of template or target DNA forced to separate from each
other during the PCR process?
3. What is a primer?
4. On which strand(s) of the template DNA do the primers anneal?
5. Why is it necessary to denature the template DNA?
6. Where do the primers anneal relative to the region to be amplified?
7. What occurs at each of the 3 temperatures in a single cycle of PCR?
a. ~95◦C
◦
b. 50-65 C
c. 70-74◦C
53
8. If you start with only one molecule of double stranded DNA template, how many
molecules of double stranded DNA are present at the end of
a. one cycle: (21) =
b. two cycles: ( ) =
c. three cycles: ( )=
d. 30 cycles: ( )=
9. How does the quantity of DNA containing target region sequence change with
each cycle?
Materials
Thermocycler
54
Thermocycler position grid sheet
For each student:

Template DNA from last session
PCR tube with a Ready-to-Go bead
For each pair of students:

P20 or P200 and P10 pipettors
Appropriate tips
A styrofoam cup with ice
For each group of four:

150 µl of Primer Mix provided in a tube on ice in a styrofoam cup
Sterile water
Permanent marker
Procedure
1. Obtain a PCR tube containing a Ready-to-Go PCR bead. This bead contains all of the
components required for PCR (other than the DNA template and the primers).
2. Use a micropipet with a fresh tip to add 22.5 microliters of PTC primer/loading dye mix to
the tube. Allow the bead to dissolve for a minute or so.
3. Use a micropipet with a fresh tip to add 2.5 microliters of your cheek cell DNA directly to
the primer/loading mix. Ensure that no cheek cell DNA remains in the tip after pipetting.
4. Store your sample on ice until your class is ready to begin thermal cycling.
5. Place your PCR tube, along with the other students’ samples, in a thermal cycler that
has been programmed to the following profile of 30 cycles.
Denaturing step: 30 seconds

Annealing step: 45 seconds
Extension step: 45 seconds
6. When the cycling program is finished, your laboratory instructor will store your samples
at -20 degrees Celsius until next week.
55
Exercise 9 (Part C): Restriction Enzyme Digestion, Gel Electrophoresis, and Phenotype Analysis
At the end of this laboratory endeavor, you should be able to:

1. Explain the purpose of restriction enzyme digestions for the purpose of identifying SNPs.
2. Explain the purpose of gel electrophoresis and explain the contribution of the gel and the current
to achieving that purpose.
3. Estimate the sizes of fragments that are analyzed by gel electrophoresis.
4. Use the terms allele, homo-, and heterozygous in explaining the results of the
electrophoresis and justify your interpretation of your results.
Introduction
In order to determine which SNP each student possesses, we can apply an enzyme called a
restriction enzyme to the PCR samples. Restriction enzymes can identify short, specific
sequences of DNA, and then cut both backbones of the double stranded DNA molecule
containing this sequence. The SNP in the TAS2R38 gene that we are studying happens to be
right in the middle of the sequence recognized by the HaeIII enzyme. Therefore, one of the
TAS2R38 gene SNP alleles can be recognized by the HaeIII enzyme (the taster SNP), while the
other one can’t (the nontaster SNP). Therefore, HaeIII will only cut PCR products from individuals
possessing taster SNPs.
In order to determine your own genotype for the TAS2R38 gene SNP, and to compare your
results with those of other members of the class, you will load two aliquots of your PCR amplified
sample (one that has been exposed to HaeIII and one that has not) alongside those of other
experimenters into wells of an agarose gel--along with the DNA size markers. Following
electrophoresis with staining, the amplification products should appear under ultraviolet light as
distinct bands in the gel. For individuals who are homozygous tasters, you should see two bands
on the gel. For individuals who are homozygous nontasters, you should see only one band on the
gel. For individuals who are heterozygous (one taster allele and one nontaster allele) you should
see three bands on the gel. See below (in this figure, U = undigested; D = digested):
56
In the process of electrophoresis, an electric current forces the migration of DNA fragments through a
special gel material. Since DNA is negatively charged, it will migrate in an electric field towards the
positive electrode (See below). The shorter a fragment of double stranded DNA is, the more quickly it can
move through the pores of the gel and migrate toward the bottom. The result is that, in the case of
double stranded DNA fragments, the fragments are separated on the basis of size.
The gel material to be used for this experiment is called agarose. When agarose granules are placed in
a buffer solution and heated to boiling, they dissolve and the solution becomes clear. A casting tray is set
up with a comb to provide a mold for the gel. The agarose is allowed to cool slightly and is then poured
into the casting tray. Within about 15 minutes, the agarose solidifies into an opaque gel having the look
and feel of coconut Jell-O. The gel, in its casting tray, is placed in a buffer chamber, connected to a
power supply, and buffer is poured into the chamber until the gel is completely submerged. The comb
can then be pulled out to form the wells into which your PCR sample will be loaded.
Loading dye is a colored, viscous liquid containing dyes (making it easy to see) and sucrose, ficoll, or
glycerol (making it dense). You will add loading dye containing ethidium bromide to your amplification
reaction and then pipet an aliquot of the mixture into one of the wells of your agarose gel. When all the
samples have been loaded, you will connect the electrophoresis chamber to the power supply. The
samples should be allowed to migrate until the blue loading dye is 1 to 2 cm from the bottom. The PCR
products run on your agarose gel are invisible to the naked eye. If you look at your gel in normal room
light, you will not be able to see the amplified products of your reaction. In order to “see” them, we
include a fluorescent dye called ethidium bromide in the gel, the electrophoresis buffer, and the loading
dye. Molecules of ethidium bromide are flat and can nestle between adjacent base pairs of double
stranded DNA (See the figure below). When this interaction occurs, the ethidium bromide molecules take
on a more ordered and regular configuration causing them to fluoresce under ultraviolet light (UV).
Exposing the gel to UV light after staining allows you to see bright, pinkish-orange bands where there is
DNA. The gel can then be photographed or captured as a digital image.
Materials – Restriction Digestion

Thermocycler set to 37 degrees Celsius for 30 minutes
For each student:

PCR product from previous exercise
For each pair of students:

P20 or P200 and P10 pipettors Appropriate tips
A styrofoam cup with ice
For each group of four:

8 microliters of HaeIII enzyme on ice
1.5 milliliter centrifuge tubes
Marker
57
Procedure – Restriction Digestion
Label a 1.5-mL tube with your assigned number and with a “U” (undigested).
Use a micropipet with a fresh tip to transfer 10 µL of your PCR product to the “U” tube. Store this
sample on ice until you are ready to begin Part IV.
Use a micropipet with a fresh tip to add 1 µL of restriction enzyme HaeIII directly into the PCR
product remaining in the PCR tube. Label this tube with a “D” (digested).
Mix and pool reagents by pulsing in a microcentrifuge or by sharply tapping the tube bottom on
the lab bench.
Place your PCR tube, along with other student samples, in a thermal cycler that has been
programmed for one cycle of the following profile. The profile may be linked to a 4°C hold
program.
Digesting step: 37°C 30 minutes
Store your sample on ice or in the freezer until you are ready to begin the electrophoresis portion.
Materials – Gel Electrophoresis

Visualization system: camera and UV light box, VersaDoc, or both UV protective
goggles or face shields if using the light box
Gloves (small, medium, large)
For each group of 4:

Absorbent paper: about 2 X 3 feet under each gel box and power supply, with room for additional
work
Power supply (may be shared between 2 groups of 4)
One 2% agarose minigel poured with 0.5 µg/mL ethidium bromide in 0.5X TBE buffer (8-10
well/gel)
Running buffer: 0.5 µg/mL ethidium brormide in 0.5X TBE
100 bp ladder in loading dye (6 µL per team of 4)
Permanent marker
For each group of 2:

P20 micropipettors and tips
Procedure – Gel Electrophoresis

1. Use a micropipete with a fresh tip to load 10 μl of the undigested (U) and 16 μl of the digested (D)
sample/loading dye mixture into different wells of a 2% agarose gel, according to the picture of the
sample gel above. Load 5 μl of the 100 bp ladder.
2. Run the gel at 130 V for approximately 30 minutes.
3. View the gel using transillumination and acquire pictures of your results.
Materials – Phenotype Analysis

Control paper strips
PTC taste paper strips
Procedure – Phenotype Analysis

1. First, place one strip of control taste paper in the center of your tongue for several seconds. Note the
taste. Then, remove the control paper, and place on strip of PTC taste paper in the center of your
tongue for several seconds. Describe the taste of the PTC paper compared to the control paper.
2. Compare your PTC genotype with your PTC phenotype.
58
Exercise 10:
Bioinformatics
1. Navigate databases at the National Center for Biotechnology Information

(NCBI) to find biological information.
2. Explain the need for searchable databases as applied to genetic testing.
Materials
Each student will require access to a laptop or tablet and the Cn3D app for windows or mac.
Instructions
Please read and follow the instructions on the following pages.
59
LESSON 2
CLASS SET
2 Navigating the
NCBI Instructions
Aim: To become familiar with the resources available at the National Center for Bioinformatics (NCBI) and the
search engine Entrez.
Instructions: Write the answers to your questions on the Student Worksheet, in your lab notebook, or on
a separate sheet of paper, as instructed by your teacher.
1. Go to the NCBI homepage http://www.ncbi.nih.gov/
List of All List of the

Resources or Most Popular
Tools Available Resources at
at the NCBI the NCBI

Figure 1: Familiarize yourself with the NCBI homepage. Credit: NCBI.
2. Take a few minutes to look around the site. The goal is to familiarize yourself with a few key components
of the NCBI.
a. What is the name of one interesting resource or database shown in the blue box on the left? What do you
think is its function or purpose? Lesson 2 – Navigating the NCBI
b. What is one interesting resource listed in the Popular Resources menu on the right? What do you think is
its function or purpose?
3. Find the search box in the center of the webpage (black box in above image). This search box uses the NCBI
search engine Entrez to look for your search term (or “query”) across all of the databases at the NCBI.
60
©Northwest Association for Biomedical Research—Updated April 2012

LESSON 2
CLASS SET
500 online books

were returned

Figure 2: Search for “BRCA1.” Credit: NCBI.
4. Type “BRCA1” into the Search box. Make sure there is no space between BRCA and 1. Click Search.
BRCA1 is a tumor suppressor gene that normally prevents cancer. Mutations in this gene are associated
with increased risk of hereditary breast cancer and ovarian cancer when normal function is lost.
The white box to the left of each database contains the number of “hits” returned from that database
(see screen shot, above). This is like searching in iTunes® without specifying categories like ringtones,
podcasts, movies, TV, or songs.
a. Why are we searching for BRCA1?
b. The Nucleotide database has DNA sequences that have been loaded onto the NCBI database.
How many times is ‘BRCA1’ cited in the Nucleotide database?
c. The PubMed database has the articles that have been published about a specific gene or disease.
How many times is ‘BRCA1’ cited in the PubMed database?
d. Compare the numbers you got for Questions a and c. Do these relative numbers surprise you?
What does this tell you about the BRCA1 gene? Explain.
Using Bioinformatics: Genetic Testing
5. Go back to the NCBI homepage by clicking the NCBI logo in the upper left corner of the screen.
This search shows that there is a lot of information at the NCBI! It can be challenging to try to make sense
of it all. Let’s start with something more familiar.
6. Click the “All Resources” link from the list of resources on the left
side of the screen.
7. Find “Map Viewer.” Click on the “Tools” tab and either scroll
through the alphabetical list, or use the “Find” feature (PC:
“Control+F” Mac: “Command+F”) to Find “Map Viewer.”
Click on the “Map Viewer” link.
The resulting page is called Map Viewer and it allows us to search

the genomes of many different organisms, including humans.
Figure 3: Click “All Resources” on the left
side of the screen. Credit: NCBI.
61

LESSON 2
CLASS SET
8. Open the Search menu, select Homo sapiens

from the pull-down menu, and click “Go.”

9. Now we can see the Homo sapiens (human) Search
genome view. A genome is all of the genetic pull-down
information in an organism. Each figure you menu
see in the “genome view” represents a pair
of chromosomes. Most of the chromosomes
are numbered, but a few are not. The
abbreviations “X” and “Y” refer to the human
sex chromosomes. Select
Homo
a. How many different types of chromosomes sapiens
do you see?
b. What does “MT” represent?

[Note: you can click the “MT” link to find out.]
Figure 4: Select “Homo sapiens” from the list of
c. With the exception of MT, the chromosomes groups or organisms. Credit: NCBI.
of the human genome are in pairs. X and
Y are a pair. Using this information and the
information from your answer to Question
9A, how many pairs of chromosomes are in
the human genome?
10. The Breast Cancer Susceptibility gene BRCA1 is on chromosome 17 in humans. [Click on the link below
chromosome 17.] Explore some of the links and views.
What do you see when you click on chromosome 17? Explore some of the links on the picture, and write
down two things you found interesting, such as the description of other genes that are also found on
chromosome 17.
11. To find the location of the BRCA1 gene, type “BRCA1” in the “Search” box at the top left of the screen,
and click “Find in This View.” Scroll through the Map of Chromosome 17 and locate the BRCA1 gene,
which should be highlighted in pink. “BRCA1” will be found in the list of Symbols. You can also use the
“Find” feature (PC: “Control+F” Mac: “Command+F”), which will highlight in yellow every mention of
“BRCA1,” including the BRCA1 gene.
Draw a picture of chromosome 17 and show the approximate location of BRCA1 on this chromosome.
Lesson 2 – Navigating the NCBI

Figure 5: Find the location of
the BRCA1 gene by using the
search function. Credit: NCBI.
62

LESSON 2
CLASS SET
12. Click on the BRCA1 link. This will take you to Entrez Gene, which provides a summary of the information
available at the NCBI for BRCA1. Scroll through the webpage and explore some of the information available.
Scroll down the webpage to the section titled “Gene Ontology.” There is a table titled “Function.”
List three of the functions that the BRCA1 protein performs.
Figure 6: Click on the “BRCA1” link to launch Entrez Gene. Credit: NCBI.

Figure 7: Scroll down to find the “Gene Ontology” section. Credit: NCBI.
Figure 8: Select “Phenotypes” from the Table of Contents. Credit: NCBI.
13. To learn about all of the phenotypes associated with mutations of BRCA1, return to the top of the web
page and from the “Table of Contents” on the right, select “Phenotypes.” This will bring you to the portion
of the web page that contains the phenotype information for BRCA1.
a. Based on what you’ve learned in class, what is a phenotype?
b. What phenotypes are associated with mutations in the BRCA1 gene? (You don’t need to click the links.)
14. Return to the Table of Contents at the top of the page and click “Reference Sequences.” This will take
you to the portion of the webpage that contains the actual genetic sequence of the BRCA1 gene.
15. Reference sequences are DNA or protein sequences that scientists, doctors and genetic counselors use
to study genes like BRCA1. You can download these sequences in different formats. For this exercise, click
“FASTA” (which is sometimes pronounced FAST-ay).
63

LESSON 2
CLASS SET
Figure 9: Click “FASTA” to obtain the FASTA sequence for BRCA1. Credit: NCBI.
Figure 10: Take a look at the FASTA sequence for BRCA1. Credit: NCBI.
16. This link takes you to the FASTA sequence for BRCA1. Scroll through the web page. This gene is very large!
a. What four letters make up this long sequence?
b. Based on what you’ve learned in class, what do these letters represent?
17. Return to the NCBI homepage by clicking on the NCBI icon on the top left of the web page.
Figure 11: Return to the NCBI homepage by clicking on the logo. Credit: NCBI.
18. Type BRCA1 in the Search box and select “Nucleotide” from the pull-down menu beside the Search box,
to limit your search to the database containing all of the DNA and RNA (Nucleotide) sequences. Click the
“Search” button.
Figure 12: Type “BRCA1” into the Search box and choose “Nucleotide” from the pull-down menu. Credit: NCBI.
64

LESSON 2
CLASS SET
19. What other organisms have BRCA1 genes? You can scroll through the list of organisms, but note that these
are listed by the scientific name of the organism (Genus and species), not the common name. For example,
Homo sapiens is the scientific name for humans. Also, the Top Organisms (or the organisms with the most
“hits”) are listed on the right. Helpful Hint: Hold your cursor over the species name to see the common name
appear. Alternatively, you can perform an internet search to find the common name(s) of your organisms.
List three organisms other than humans that have BRCA1 genes. Include both the scientific and common names.
Figure 13: Scroll through the list of Top Organisms. Credit: NCBI.
20. Look back at your list of functions for the BRCA1 gene (question #12).
Does it surprise you that so many organisms share the BRCA1 gene? Explain.
21. What kind of information can you find at the National Center for Biotechnology Information?
Summarize what you have learned today by listing three types of information found at the NCBI.
65

LESSON 4
CLASS SET
4 Instructions for Aligning Sequences

with BLAST
Aim: To understand how genetic tests are performed by comparing the DNA and protein sequences of a family
to determine who is carrying the BRCA1 mutation.
BLAST — Basic Local Alignment Search Tool
Background on BRCA1
For cells to function properly, they need to be able to repair errors in their DNA. These errors can arise when DNA
is being copied, or when DNA somehow becomes damaged when exposed to chemicals or radiation. The breast
cancer susceptibility gene (BRCA1) encodes a protein that is involved in DNA repair. When a DNA strand is broken,
the BRCA1 protein works with other proteins to help repair the break. If these breaks are not repaired, the DNA
damage can ultimately lead to cancer. Therefore, BRCA1 is known as a tumor suppressor, because it helps prevent
the formation of tumors (which can arise when DNA errors go unrepaired). Mutations to the BRCA1 gene can
interfere with or abolish the BRCA1 protein’s normal function, thus allowing cancer to develop.
Instructions: Write the answers to your questions on the Student Handout—Aligning Sequences with BLAST
Worksheet in your lab notebook or on a separate sheet of paper, as instructed by your teacher.
PART I: Aligning DNA Sequences to a Reference Sequence
Lesson 4 – Understanding Genetic Tests to Detect BRCA1 Mutations

1. Access the DNA sequence file your teacher has given you. It contains the BRCA1 reference DNA sequence and
six individual Lawler family sequences.
2. Go to the NCBI blast website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
3. Select “nucleotide blast,” as shown in Figure 1, since we will be comparing a DNA sequence (sequence of
nucleotides) to a DNA sequence (sequence of nucleotides). Note that there are options for comparing protein
sequences to protein sequences, and others.
Figure 1: Select “Nucleotide BLAST.”

Credit: NCBI BLAST.
66

LESSON 4
CLASS SET
4. From the nucleotide blast page, click the box to choose the option to “Align two or more sequences”
(see Figure 2).
Figure 2: Align two or more

sequences. Credit: NCBI BLAST.

5. A second text box will appear.
6. Copy the reference sequence for BRCA1 from the file, including the “>” (“caret”) symbol and the
name, and paste it into the top text box (see Figure 3).
Figure 3: Copy the BRCA1

reference sequence.
Credit: NCBI BLAST.
7. Copy the DNA sequence from the person you are testing (Deb, Lori, Katherine, Mother, Father, or Uncle)
and paste it into the bottom text box. Again, include the “>” symbol and the name.
8. Click “BLAST.”
9. When your search is complete, you will see a window with the BLAST results, showing an alignment of
the two DNA sequences you entered above.
10. Click the “Formatting Options” link located near the top of the page (see Figure 4).
Figure 4: Click the “Formatting Options” link.

Credit: NCBI BLAST.
67

LESSON 4
CLASS SET
11. Find the Alignment View and use the drop-down menu to choose “Query-anchored with dots for identities.”
The query is the reference sequence. The query-anchored view shows the reference sequence at the top with
the subject sequence aligned below (i.e., the family member’s sequence or a patient’s sequence). Dots are
used to show nucleotides that are identical and letters are used to show nucleotides that differ.
12. Click the “Reformat” button (see Figure 5).
Figure 5: Click the “Reformat” button.

Credit: NCBI BLAST.
13. Scroll down the page to see if there are positions where the query sequence (which is the reference
sequence) differs from the subject (family member’s or patient’s) sequence. In other words, look for a place

where there is a letter instead of a dot, showing that there’s been a change in the nucleotide at that position.
Note the numbers at the ends of the lines refer to the position of the nucleotide (see Figure 6).
Reference sequence
Family member’s sequence
Nucleotide Nucleotide Nucleotide

#1 #10 #60
Figure 6: Comparing the query and subject sequences.

Credit: NCBI BLAST.
14. Explain why you think we need to compare DNA sequences and protein sequences of people in this
family to a “reference sequence.”
15. Are there any differences between the reference sequence (the top sequence marked “query”) and the
sequence you entered? What do you think this means?
68

LESSON 4
CLASS SET
16. BLAST is a powerful tool that can align more than one sequence at a time. Scroll to the top of the page
and click the “Home” button (see Figure 7).
Figure 7: Click the “Home” button.

Credit: NCBI BLAST.
17. Go back to Step #2 to start a new BLAST alignment. This new alignment will include sequences from the
entire Lawler family.
18. Copy the reference sequence for BRCA1 from the file, including the “>” symbol and the name, and
paste it into the top text box.
19. Copy the DNA sequences from all six individuals (Deb, Lori, Katherine, Mother, Father, and Uncle) and
paste them into the bottom text box. Again, include the “>” symbols and the names. This can be done in
one copy and paste function from the DNA Sequence File.
21. Scroll down the page to see if there are positions where the query (reference) sequence differs from the
subject (family member) sequence. Note that the numbers at the ends of the lines that refer to the position
of the nucleotides, as shown in Figure 8.

Figure 8: Note the numbers that refer to the positions of the nucleotides.
Credit: NCBI BLAST.
22. In the box above the alignment, you can see the legend for the sequence ID and the name of the subjects,
as shown in Figure 9.
Figure 9: Sequence ID
and subject names.
Credit: NCBI BLAST.
69

LESSON 4
CLASS SET
23. Do all of the family members have the same mutation? What is the location of the nucleotide that differs in some of
the family members? (You will need to look at the numbers on the side of the alignment.)
24. On Student Handout—Aligning Sequences with BLAST Worksheet, circle the names of the Lawler family
members who have this mutation, or list the names in your lab notebook or on your homework paper.
25. These differences, or changes to the DNA sequence, represent a mutation to the BRCA1 gene. However,
we need more information to determine whether this mutation results in a change in the amino acid found
in the BRCA1 protein. Amino acids are encoded by three bases, called a codon. On Student Handout—
Aligning Sequences with BLAST Worksheet, complete the table, including the codons and resulting amino
acids (as represented by a one-letter abbreviation), or create a similar table in your lab notebook or on your
homework paper. See the codon table as instructed by your teacher.
Lawler Family Sequence Analysis
Reference Sequence Mutated Sequence

DNA Coding Strand ATG ?
DNA Template Strand TAC ?
mRNA Codon AUG ?
Amino Acid ? ?
26. What does it mean for the individual if that person has the mutation?

27. What does it mean for the individual if that person is free from the mutation?
28. Record your results from the nucleotide BLAST alignment in your BLAST Results Document by capturing
an image from your computer screen using the following instructions:
a. Open up a new Word® document and label the document with your LASTNAME_BRCA1_NCBI. Type your
name, class period, and date at the top of the blank page and add the title “BLAST Results Document.”
b. Return to the BLAST results page.
c. Scroll down until the sequence showing the BRCA1 mutation is centered on the computer screen.
i. For PC users: Hit the Prnt Scrn button on your keyboard. This is often on the top right of the
keyboard, to the right of the F12 button.
ii. For Mac users: Press the keys: Command + Shift + 4 at the same time. The image will be saved on
your desktop.
d. Return to your Word® document.
i. For PC users: Use the paste function to transfer the captured image (Crtl + V).
ii. For Mac users: Open the Insert menu, choose Picture from file, and choose the image that you
captured from the computer screen.
e. Save this document. Transfer it to a thumb drive or email it to yourself if you will not have access to this
computer in the future.
70

LESSON 4
CLASS SET
PART II: Aligning Protein Sequences to a Reference Sequence

29. Access the Protein sequence file your
teacher has given you. It contains the

BRCA1 reference protein sequence and six
individual Lawler family sequences.
30. Go back to the NCBI blast website

(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
31. Select “protein blast” (as shown in

Figure 10) since we will be comparing
a protein sequence (sequence of amino
acids) to a protein sequence (sequence of
amino acids).
32. From the protein blast page, click

the box to choose the option to “Align
two or more sequences,” as shown in
Figure 11.
Figure 10: Select “Protein BLAST.”
Credit: NCBI BLAST.
Figure 11: Select “Align two

or more sequences.”
Credit: NCBI BLAST.
33. A second text box will appear.
34. Copy the reference sequence for BRCA1 from the file, including the “>” symbol and the name, and paste it
into the top text box.
35. Copy all six protein sequences from the entire Lawler family (Deb, Lori, Katherine, Mother, Father, and
Uncle) and paste them into the bottom text box. Again, include the “>” symbol and the name.
37. When your search is complete, you will see a window with the BLAST results, an alignment of all the protein
sequences you entered above.
71

LESSON 4
CLASS SET
38. Click the “Formatting options” link located near the top of the page, as shown in Figure 12.
Figure 12: Click “Formatting options.”

Credit: NCBI BLAST.
39. Find the Alignment View (see Figure 13) and use the drop-down menu to choose “Query-anchored with
dots for identities.” Dots are used to show amino acids that are identical and letters are used to show
the amino acids that differ.
40. Click the “Reformat” button as shown in Figure 13.

Figure 13: Locate “Alignment View” and the “Reformat” button.
Credit: NCBI BLAST.
41. Scroll down the page to see if there are positions where the query (reference) sequence differs from the
subject (family member’s or patient’s) sequence. In other words, look for a place where is there a letter instead
of a dot, showing that there’s been a change in the amino acid at that position. Note that the numbers at the
ends of the lines refer to the position of the amino acid, as shown in Figure 14.
Reference sequence
Family member 1
Family member 2
Family member 3
Family member 4
Family member 5
Family member 6
Amino Acid Amino Acid Amino Acid

#1681 #1701 #1740
Figure 14: Compare the reference and subject sequences.

Credit: NCBI BLAST.
72

LESSON 4
CLASS SET
42. Are there any differences between the reference (query) sequence and the family member sequences you
entered? If so, do all of the family members have the same mutation? What was it?
43. These differences, or changes to the amino acid sequence, are a result of the mutation in the BRCA1 gene.
Answer the following questions:
a. What is the amino acid in the reference sequence (as represented by a one-letter abbreviation)?
b. What is the amino acid in the sequences containing the mutation?
c. Is this the amino acid that you expected based on your DNA analysis in Part I, in the table “Lawler Family
Sequence Analysis?”
d. Where is the location of the change? (Count carefully using the reference numbers at the end of the row.)
44. Which individuals in the Lawler family have the change in their amino acids?
45. Are your answers to Question #44 the same as your answers from your DNA analysis (Part I, Question #24)?
Is this what you would expect? Why or why not?
46. Record your results from the protein BLAST alignment by capturing an image from your computer and saving
it in your BLAST Results Document from Step #28. Refer to the image capturing instructions in Step #28 if
you need help with this step.
47. Now that you have some test results, return to Student Handout—Lawler Family Pedigree.
i. Fill out as much additional information as you can for the pedigree.
ii. Which Punnett square most accurately represents the Lawler family? Why?
PART III: Extension Questions
48. M is the one-letter code for the amino acid methionine. R is the one-letter code for the amino acid
Arginine. Look at the structures below.
METHIONINE (M) ARGININE (R)

Neutral, Non-Polar Positive Charge, Polar
49. Describe at least three ways in which methionine and arginine are different (i.e., polarity, size, shape, composition).
50. How might this mutation affect the protein?
73

LESSON 5
CLASS SET
5 Instructions for Seeing DNA in 3D

Aim: To understand that protein structure can impact protein function, using the bioinformatics tool Cn3D
to visualize molecules.
Instructions: Write the answers to your questions on Student Handout—Seeing DNA in 3D Worksheet,
in your lab notebook, or on a separate sheet of paper, as instructed by your teacher.
PART I: Viewing DNA Structure
1. Go to the NCBI Structure database: (http://www.ncbi.nih.gov/structure).
2. The center top of the page has an open area for search terms. Type 1NAJ into the search area and click
the Go button.
We will begin our investigation of 3D structure with a look at a molecule you are already familiar with:
DNA. “1NAJ” is the accession number for a file that contains structure information for a small piece of
double-stranded DNA. An accession number is like a catalog number or bar code; it bears no resemblance
to the product itself, but allows you to access information in the vast databases at the NCBI.
3. The 1NAJ record will appear as the only result from this search. Click the DNA image that looks like two
colored strings winding loosely around each other with a black background.
You are on the Structure Summary page, which includes a brief description of the source of the structure.
4. Click the “View structure” button to download the

structure file, as shown in Figure 1. Save this file to
Lesson 5 – Learning to Use Cn3D: A Bioinformatics Tool

your desktop, or to a folder, as instructed by your
teacher. Then double-click on the file to open it.
[Note: The file may appear in different places

depending on the type of web browser and how the
computer has been set up. You may need to look for
your file in your Downloads folder. This file may have a
name like “mmdb.cgi.”]
5. If your file opens with the Cn3D program, skip to

Step #7. Otherwise, find the Cn3D program icon on
your desktop or in your program menu, and open the
program. Select “Open” from the file menu and select
Figure 1: Click the “View structure”
your file from above.
button to download the file.
[Note: The Structure Window is the black square box
that shows the DNA structure. Your screen may also
show the Sequence/Alignment viewer, below the
Structure Window.]
74
©Northwest Association for Biomedical Research—Updated January 27, 2012

LESSON 5
CLASS SET
6. View the DNA in the Structure Window. Drag the structure with your mouse to turn it around. Hold the
shift button down, and continue to hold it down, while dragging, to move your structure without turning
it. Experiment with controlling the movement and viewing the molecule from multiple angles.
You can zoom in and out by using the z or x keys. Alternatively, you can change the size by holding down
the left mouse button and dragging the mouse away from the screen.
Hold down the left mouse button and drag the mouse towards or away from the screen.
7. Open the View menu and choose “Animation.” Choose the “Spin” option.
What happens when you click s and n?
Position the DNA molecule the way you want it before proceeding.
8. Open the Style menu and select “Rendering Shortcuts.” There are a variety of rendering options
available to view the DNA molecule. Explore each of the following:
Worms Tubes Wire Ball and Stick Space-fill
Imagine you are teaching a class about DNA. What rendering option(s) would you use to teach students
about DNA structure? Explain the reasons for your decision.
9. Open the Style menu and select “Coloring Shortcuts.” There are many options for changing the colors
of molecules. Select each of the options below, and answer each of the following questions.
a. Object: How did this change the coloring of the DNA molecule?
b. Rainbow: ‘Rainbow’ uses the color red at the start (5’ end) and continues through the rainbow.
Why are there two red regions?
c. Explore a few combinations using different rendering and coloring options. Which coloring option do
you find most useful? Why?
10. Go to the Sequence/Alignment Viewer window on your screen.
[Note: The Sequence/Alignment Viewer is the window below the Structure Window that shows the
sequence being viewed, as shown in Figure 2. If there is no sequence visible, open the Window menu
and choose “Show Sequence Viewer.”]
Click on the letter of a

base in the Sequence/
Alignment Viewer and

see what happens.
Structure
Note that there are two Window
sequences: 1NAJ_A and
1NAJ_B. Each sequence
corresponds to a different
strand of the DNA
Sequence/
molecule. Alignment
Viewer
Letters of
bases

Figure 2: The Sequence/Alignment Viewer shows the sequences being viewed.
75 Credit: Wu et al. 2003.

LESSON 5
CLASS SET
11. In the sequence 1NAJ_A, click the first “g.” Next, click the first “g” of the sequence 1NAJ_B.
Where are these two guanines located on the DNA molecule? Explain why.
12. Choose a single base you would like to view, and click on it in the Sequence/Alignment viewer.
13. Change the Rendering style to “Wire” and the Coloring style to “Object.”
a. What does that do? Explain.
b. Move the DNA so that the ring structure(s) of the selected nucleotide’s base is clearly seen.
What do you see?
14. Open the Select menu and choose “Show Selected Residues.” Answer the following questions:
a. What do you see?
Zoom in to view the base more easily (refer to Step #7 if you need help).
b. Challenge question: Is the base you selected a purine or a pyrimidine, and how do you know?
From the Style menu choose “Show Everything” to see the complete strand.
PART II: Viewing the BRCA1 Protein
Now that you are familiar with the Cn3D program, we will view part of the BRCA1 protein and part of a
second protein that it interacts with.
15. Go to the NCBI Structure database: (http://www.ncbi.nih.gov/structure) or click the “Structure Home”
button from the DNA structure summary page.
16. At the top of the page, find the search option. Type “1Y98” into the blank and click the “Go” button.

17. The 1Y98 record will appear as the only result from this search. Click on the link or title of the structure
above the black box with the picture of the protein in it. You are on the Structure Summary page, which
includes a brief description of the source of the structure.
18. Click the “View structure” button to download and open the structure file.
19. Take a moment to explore different Style options.
20. Open the Style menu and choose “Coloring Shortcuts ÚSecondary Structure” to highlight each type
of secondary structure in a different color.
21. Look at the Sequence/Alignment Viewer below the structure. When viewing the DNA structure, the
Alignment/Sequence Viewer showed the letters of the bases. When viewing a protein structure, the viewer
shows the one-letter abbreviation for the amino acids.
Note that colors of the amino acid sequence correspond to the colors of the structure.
22. The structure contains two BRCT domains. To see each domain individually, open the Style menu and
choose “Coloring Shortcuts ÚDomains.”
This will show the two BRCT domains in different colors. BRCT stands for Breast cancer C-Terminal
domain, and is involved in protein-protein interactions. The alpha helices are depicted as cylinders (like
crayons) and the beta sheets are depicted as flat arrows. Both the helix and strand objects point in an
amino to carboxyl direction (i.e., they point toward the end of the protein). 76

LESSON 5
CLASS SET
Note that there is a third domain – this is part of another, different protein to which BRCA1 normally binds
to do its job of repairing DNA. This protein is called CtIP.
23. Find the portion of the second protein, CtIP, that BRCA1 interacts with during DNA damage repair.
It looks like a little brown squiggle off to the side. In the Sequence Window, the sequence of this portion
of the second protein is labeled 1Y98B. If you can’t find CtIP, highlight the 1Y98B sequence and the
CtIP will light up yellow in the Structure Viewer. To remove the yellow highlights, click somewhere the
Sequence Window to activate the sequence menu. Then, open the View menu and choose “Clear
Highlights.”
24. Now that you know what the portion of the CtIP protein looks like, go back to the Style menu and
select “Coloring Shortcuts Ú Secondary Structure.” Move your cursor along the 1Y98A amino acid
sequence in the Sequence/Alignment Viewer, as seen in Figure 3.
Note that the colors of the amino acids correspond to the colors in the protein structure (beta sheets,
alpha helices, and random coils). The box on the lower left of the screen shows the location of the amino
acid your cursor is over.
a. What color are the beta sheets?
b. What color are the alpha helices?
25. Move your cursor along the amino acid

sequence until you get to amino acid
1Y98A_loc130 (PDB 1775).
Amino acid 1775 (PDB 1775) is the

location of the M1775R mutation in
BRCA1 that affects the Lawler family.
Amino acid color corresponds

to color in structure.
Slide the scroll bar to see more

of the amino acid sequence.
The location number (PDB) appears

when the cursor is over an amino acid.

Figure 3: When you open the BRCA1 structure, you will see the BRCA1
protein that does not contain any mutations.
Credit: Varma et al. 2005.
77

LESSON 5
CLASS SET
26. Click on amino acid number “1775” (M, the amino acid methionine) in the Sequence Viewer to highlight
it in the structure.
It should light up yellow in the structure. You are looking at where the mutation would be located, but this
structure you are looking at does not have the mutation. (Recall that the mutation converts a M to a R.)
27. Open the Select menu Ú Select by Distance. Open the Select menu Ú Select by Distance Ú
Residues only. Leave the distance cutoff at five Angstroms to select all molecules within a radius of
five Angstroms. Make sure the options “Select protein residues” and “Select other molecules only” are
checked. Click “OK.”
[Note: An Angstrom, Å, is 1x10-10 meters.]
28. Note that part of the CtIP protein now also lights up yellow, as do other BRCA1 amino acids (residues). It
is very close to the site on BRCA1 where the mutation would be. You can imagine how a change in BRCA1
might impact its ability to interact with this second protein, CtIP, which is also required for DNA repair.
29. Record your results from Cn3D by capturing the image using the following instructions:
a. Open up the Word® document you created in Lesson Four. It should be labeled with your LASTNAME_
BRCA1_NCBI.
b. Return to the 1Y98A Cn3D page with the protein structure.
c. FOR PC USERS: Hit the “Prnt Scrn” button on your computer. This is often in the top right of the
keyboard, to the right of the F12 button.
FOR MAC USERS: Press the keys: “Command + Shift + 4” at the same time.
d. Return to your Word® document.
e. FOR PC USERS: Use the paste function to transfer the screen shot.
FOR MAC USERS: Open the Insert menu, choose “Picture from file,” and choose the image that
you captured from the computer screen.

[Note: The image may have saved to your desktop.]
f. Type these instructions and questions at the bottom of your Word® document:
1. Circle the location of the mutation in the picture of the BLAST alignment.
2. Circle the location of the mutation in the Cn3D picture of the protein structure.
3. Explain what these pictures represent.
4. Explain how the pictures are connected to each other.
g. Follow the instructions you typed above and answer the questions on your Word® document. You may
choose to print out the document later and circle the points of mutation, or you can make the circles
directly on your Word® document by doing the following:
Use your drawing tools in Word® to draw red circles around the mutation in the BLAST sequence
window and the Cn3D view. (Insert Shapes Ú Oval Ú Shape Fill: no fill, Shape outline: red line).
You can also use the “Crop” feature in Word® to crop your image.
h. Save and close this document. Transfer it to a thumb drive or email it to yourself if you will not have
access to this computer in the future.
78

LESSON 5
CLASS SET
30. Challenge: To get a better sense of the interaction between BRCA1 and CtIP, Cn3D can show the amino
acids that are interacting with each other in ball and stick form. Be sure that the amino acids in the
previous step are highlighted before you begin.
a. Open the Style menu and choose “Annotate Ú.” Click on the box that says “New” to the right of
“Selection: (new).” Another small Edit Annotation window will open.
b. Click on “Edit Style” in the Edit Annotation window. There is no need to enter a description. A big
Style Options window will open and will default to the “Settings” tab.
c. Find the group Protein Sidechains, and click on the box next to it to check it.
d. Immediately to the right, pull down the “Rendering Options for Protein Sidechains” and change
them to “Ball and Stick.”
e. Click “Apply” and “Done” (and “OK” and “Done” on the remaining windows) to close all the
windows and return to the structure.
f. You may want to capture this image for your Word® document as well.
g. Challenge Question: What can you see now that you could not see before you annotated this structure?
Does this help you understand the consequences of the Lawlers’ M1775R mutation?
31. Optional: If time permits, you may wish to experiment with protein structures 1JNX and 1N5O. Make
sure that you enter capital O and not a zero for “1N5O.”
1JNX: This is the accession number for the BRCA domains of the non-mutated version of the BRCA1 protein.
1N5O: This is the accession number for the BRCA domains of the M1775R mutation of the BRCA1 protein.
79

LESSON 5
HANDOUT
Name ____________________________________________ Date _____________ Period _________
5 Seeing DNA in 3D
Worksheet
Aim: To understand that protein structure can impact protein function, using the bioinformatics tool Cn3D
to visualize molecules.
Instructions: Use Student Handout—Instructions for Seeing DNA in 3D to complete this worksheet.
PART I: Viewing DNA Structure
7. What happens when you click s and n?
s:
n:
8. There are many ways to view the DNA. Imagine you are teaching a class about DNA. What rendering
option(s) would you use to teach students about DNA structure?
(circle one): Worms Tubes Wire Ball and Stick Space-fill
Explain the reasons for your decision:

9a. Look at Coloring Shortcuts, and click “Object.” How did this change the coloring of the DNA molecules?
9b. Now look at Rainbow: “Rainbow” uses the color red at the start (5’ end) and continues through the
rainbow. Why are there two red regions of the DNA?
9c. Which coloring option do you find most useful? Why?
80

LESSON 5
HANDOUT
11. When you click on the two guanines that are in the same place for both DNA strands, where are they
located on the DNA molecule? Explain why.
13a. After changing the Rendering style to “Wire” and the Coloring style to “Object,” what does that do?
Explain.
13b. Move the DNA so that the ring structure(s) of the selected nucleotide’s base is clearly seen. What do you see?
14a. After selecting “Show Selected Residues,” what do you see?
14b. Challenge question: Is the base you selected a purine or a pyrimidine, and how do you know?
PART II: Viewing the BRCA1 Protein
24a. What color are the beta sheets?
24b. What color are the alpha helices?

30. Challenge Question: What can you see now that you could not see before you annotated this
structure? Does this help you understand the consequences of the Lawlers’ M1775R mutation?
81

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Biology 1100 Lab Manual

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Department of Biological Sciences

Exercise 1: Micropipeting Basics .............................................................................................................. 3

After completing this laboratory topic, you should be able to:

Pipeting is a critically important technique in life science experiments to ensure accurate

Figure 1.1. Microcentrifuge tubes.

Figure 1.2. Single-channel variable Figure 1.3. Multi-channel variable

Dyes (Red, Blue, Yellow)

Measuring Small Volumes with Micropipettes

Typical Micropipette Labels and Ranges

Figure 1.4. Sample delivery with micropipets.

Micropipetting Using a Variable Volume Micropipette

In this activity you will:

After completing this laboratory topic, you should be able to:

1. Compare and contrast different types of microscopes.

Per group of two students:

What are the Different Types of Microscopes?

Getting to Know the Microscope

Before working with the

E. How Can You Adjust the Light

Pre Lab Questions

1. What is the difference between magnification and resolving power?

2. Which objective do you use to locate specimens on your slide? _________________________

Carefully re-center the e on the slide and switch to the low

Carefully re-center the "e" again and switch to the high

Use the fine adjustment knob only to bring the specimen

Draw the letter “e” to scale. The circles below represent

2. What happens to the working distance as you change objectives?

Part 2: What is the Field of View and How Can It Be Measured?

High Power Field of View = _______

1. Check that each section of this page is complete. ✅

A. Making a Wet Mount of a Slide

Place one drop of stain (methylene blue or iodine) on

C. Viewing Large Specimens and Dissecting

Name/Description of specimen(s) viewed: _________________________________________________

Which color thread is on top? _____________ middle? ______________ bottom?

After completing this laboratory topic, you should be able to:

Maintenance of the steady state of a cell is achieved only through regulated

30 cm strip of moist dialysis

Dialysis tubing is a membrane made of regenerated cellulose fibers

You will use two tests in your experiment:

Figure 3.3. Experimental setup for Experiment 3A.

2 compound microscopes labeled A and B

For this experiment, two slides have been set up on demonstration

Solution Contents Original Final Color Glucose

2. How can you explain your results?

3. From your results, predict the size of I2KI molecules relative to

Which solution is hypotonic?

2. Which solution has the greatest osmolarity?

3. Would you expect pond water to be isotonic, hypertonic or hypotonic to

4. Verify your conclusions with your laboratory instructor.

1. Complete Table 3.2 and provide a title. To calculate percentage change in

Weight change = final weight – initial weight

Percentage change in weight = (weight change/initial weight) X 100

If the sample increased in weight, the value should be positive. If it

Discussion (to be completed next week)

2. Explain how this information can be used to determine the osmolarity of

3. Estimate the osmolarity of the potato tuber tissue.

After completing this laboratory topic, you should be able to:

1. Understand the process of alcoholic fermentation in yeast, and under

Alcohol Fermentation in Yeast

For centuries, humans have taken advantage of yeast fermentation to produce

4 respirometers: test tubes, 1 ml graduated pipettes, aquarium tubing, flasks,

Which color thread is on top? _ middle? __ bottom?