Biology Connection: Appendix 2: Examples of Interdisciplinary Chemistry-Biology Laboratory Experiments
Biology Connection: Appendix 2: Examples of Interdisciplinary Chemistry-Biology Laboratory Experiments
Biology Connection: Appendix 2: Examples of Interdisciplinary Chemistry-Biology Laboratory Experiments
CHEM 1100
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CHEM 1100
History of Soap
The discovery of soap dates back to about 6000 years ago. Around 2800 B.C.E, the ancient Babylon
excavations uncovered cylinders with inscriptions for making soap.1 In 1500 B.C.E, records from ancient
Egypt described how animal and vegetable oils were combined with alkaline salts to make soap.
According to a Roman legend, “soap got its name from Mount Sapo, where animals were sacrificed. Rain
washed the fat from the sacrificed animals along with alkaline wooden ashes from the sacrificial fires into
the Tiber River, where people found the mixture helpful in cleaning clothes. This procedure for making
soap remained unchanged for centuries, with American colonists collecting and cooking down animal
tallow (rendered fat) and then mixing it with an alkali potash solution obtained from the accumulated
hardwood ashes of their winter fires. Similarly, Europeans made castile soap using olive oil. Since the
mid-nineteenth century, the process became commercialized and soap became widely available at the
local markets.”1 To date, most people use similar methods to make home-made soaps.
Soap making involves the hydrolysis of a triglyceride (fat or oil) using an alkaline solution usually lye,
chemical name sodium hydroxide. Triglycerides are typically triesters consisting of 3 long-chain
aliphatic carboxylic acid chains appended to a single glycerol molecule (see Equation 1). This process of
making soap is known as saponification. The common procedure involves heating animal fat or
vegetable oil in lye (sodium hydroxide), therefore hydrolyzing it into carboxylate salts (from the
combination of carboxylic acid chains with the cations of the hydroxide compound) and glycerol.
Equation 1:
H2C O C R
CH2 OH O
O
CH2 OH +3H O C R
CH O C R
CH2 OH
O
Fatty acid chain R
H2C O C
Glycerol
A triglyceride
Equation 2 shows the general reaction between triglycerides and sodium hydroxide, while Equation 3
shows an example of a specific reaction between beef fat and sodium hydroxide to form soap. 1, 2 Notice
that from these reactions, I mole of triglyceride requires 3 moles of NaOH to produce I mole of glycerol
and 3 molecules of soap. Soap has unique properties that make it an excellent surfactant (“surface
active” compound) or cleaning reagent. In this lab, you will: (1) synthesis soap, and (ii) study the physical
and chemical properties of your soap.
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CHEM 1100
Equation 2:
O
O
H2C O C R' O- Na+
R' C
CH2 OH O
O H2O/Ethanol
CH O C R'' CH2 OH + R'' C O- Na+
NaOH
O
O Heat CH2 OH
R'" R'" C O- Na+
H2C O C 1
Eqn 2: General model showing the hydrolysis of triglycerides with sodium hydroxide
Equation 3:
O
O
H2C O C C17H25 O- Na+
C17H25 C
CH2 OH O
O NaOH
CH O C C17H25 CH2 OH +
-
C O Na
+
Heat C17H25
O
O CH2 OH
C17H25 C17H25 C O- Na+
H2C O C
Glycerol Sodium stearate
Stereol-a triglyceride (Soap)
(beef fat)
Questions to ponder:
What are the differences between cooking fat and cooking oil?
Do you think different fats or oils will produce the same type of soap in terms of color, smell, and
texture? (Share your ideas with your group members, then with the rest of the class).
A. Synthesis of Soap
Experimental Procedure
Obtain triglyceride (cooking fat/oil) from your TA, and place about 6 mL of the cooking oil or 6g of
cooking fat into a 120 mL Erlenmeyer flask. Dissolve 5 g of NaOH into 40 mL of 50/50 water- 95%
ethanol (CAUTION: NaOH is very corrosive and may result into severe burns. If the chemical
comes into contact with your skin, wash the affected area with copious amount of water). Add the
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CHEM 1100
NaOH solution into the 120 mL flask containing the oil, swirl the solution gently. Clamp the flask and
submerge it in a 250 mL beaker of boiling water. Heat the solution for 45 minutes. (CAUTION: Be
careful with the hot NaOH. Do not look into the mouth of the flask. If splattering of the mixture
occurs, remove or lower the heat). While allowing for the stated reaction time, prepare a salt solution of
25 g of NaCl in 150 mL of distilled water in a 250 mL beaker. Place the beaker in an ice-water mixture to
cool the salt solution. (Also, brainstorm about the questions on section B, and prepare solutions for
part B (II) as you wait for your soap to form). Pour the soap solution into the cooled salt solution after
the reaction time is complete (i.e., after 45 minutes) and stir for several minutes. Filter the precipitated
soap through vacuum filtration (as directed by the TA). Wash the solid (soap) in the funnel at least
thrice with a10 mL ice-cold water each time. Weigh 3 g of your soap and preserve for part B. Place the
remaining soap into a paper cup to harden.
Discussion questions
1. Why is it important to cool the salt solution before adding the soap solution?
2. What is the smell of your soap (record the smell in the data sheet A)? How does the soap smell
differ from the smell of the triglyceride used?
3. What is the color of your soap (record your observation in the data sheet A)? Does the color of
your soap differ from those of your classmates? If so, why?
4. What texture is your soap? Does you soap have same texture as those of your peers in other
groups? If so, why?
In part A, you have synthesized soap and studied some physical properties such as the soap color, soap
smell, and soap texture. In this section, you will investigate the chemical properties of soap. You will also
compare the chemical properties of your soap with the detergent provided by your instructor. How is this
possible? Think about how you can design an experiment to study the chemical properties of soap and
detergent, and share your ideas with your group partners, then with the rest of the class. How do your
ideas compare or differ from the other groups’ ideas.
Experimental Procedure
Label two clean dry 150 mL beakers; one ‘soap’ and the other ‘detergent’. Dissolve the 3 g of soap
preserved in part A in 100 mL of boiling water in the beaker labeled ‘soap’ and 3 g of detergent provided
in 100 mL of boiling water in the beaker labeled ‘detergent’. Preserve these solutions for the subsequent
sections. You are required to share your ideas with your group members on how you can test for the
chemical properties of soap and detergent using the reagents/materials provided.
In your group, discuss how you can determine the pH of your soap and the detergent using a pH
paper. Make sure you get approval of your test method from the TA before carrying out the test.
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CHEM 1100
What is the pH value for your soap and the detergent? Specify if acidic, neutral, or basic and record
your observations and inferences on the data sheet B provided.
Using the reagents provided, devise a procedure with your group members to determine the behavior of
your soap and the detergent provided with the reagents below (make sure your TA approves your
method before proceeding with the test). Record your observations in data sheet B and account for the
observed phenomena as much as possible.
3 mL distilled water
3 mL distilled water + 5 drops 3% calcium chloride solution
3 mL distilled water + 5 drops 3% magnesium chloride solution
3 mL distilled water + 5 drops 3% iron (III) chloride solution
3 mL distilled water + 5 drops 3% sodium chloride solution
3 mL distilled water + 5 drops 3% ammonium sulfate solution
III. Testing for the solubility of soap and detergent in acidic water
Earlier in the semester, you carried out solubility tests on some reagents. Using the knowledge acquired
from that lab, how can you devise a procedure to test for the solubility of soap and detergent in 1 M HCl
solution? Present your ideas to the TA for approval before carrying out your test. Record your
observations in data sheet B and account for the observed phenomena (obtain 1M HCl solution for TA).
IV. Testing for the emulsifying power of soap, detergent, and distilled water on mineral oil
In this test, you will investigate the behavior (reaction) of each reagent (soap, detergent, and distilled
water) with mineral oil. Devise a method to set-up the experiment and get approval from your TA
before proceeding with the test. Record your observations in data sheet B and account for the observed
phenomena.
References
1. http://www.chemistryexplained.com/Ru-Sp/Soap.html.
2. Dueno et al., Journal of Chemical Education Vol 75(5) 1998.
3. C. E. Harland, Ion exchange: Theory and Practice, The Royal Society of Chemistry, Cambridge,
1994.
4. Muraviev. D., Gorshkov, V., Warshawsky), Dekker, M. (2000). Ion exchange, New York.
5. http://chemistry.about.com/od/cleanerchemistry/a/how-soap-cleans.htm
6. David A. Katz (2000). The science of soaps and detergents.
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CHEM 1100
Post-Lab Questions
1. Using the structure of the triglyceride (Canola oil), deduce the structure of the soap (show your
reaction equations).
3. Explain the reason(s) behind the observed difference in texture between the Crisco fat and the
cooking oils.
4. Based on your findings, which reagent (soap or detergent) lathered well with hard water? Why?
6. Which solutions in part B (III) formed scum, and which ones formed suds? Account for the observed
phenomena.
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Lab 7i
Polymerase Chain Reaction
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Introduction
For thousands of years, farmers have selectively bred crops such as corn and
soy beans to produce desirable qualities like higher yields, greater insect resistance,
better nutrition and/or being more resistant to drought. However, this kind of selective
breeding takes time and crop character manipulation can sometimes be unpredictable,
as increasing one quality sometimes has the unintended consequences of decreasing
another.
Advances in molecular biology have sped up the pace of these changes by
allowing the insertion of genes or fragments of genes from other organisms that have
already evolved their genes to handle these problems. This recombinant DNA revolution
has produced improved crops with increased resistance to insects and therefore allows
farmers to use fewer pesticides. Other changes to crops have provided crops with
higher nutritional value and the ability to grow in areas where the conditions were not
sufficient previously.
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PCR Technique
We will be using the
Polymerase Chain Reaction
(figure 1) in today’s lab to detect
the presence of transgenes in
tortilla chips. An excellent video of
the technique can be found at
http://youtu.be/eEcy9k_KsDI.
The starting material for
PCR, the 'target sequence,' is a
gene or segment of DNA. In a
matter of hours, this target
sequence can be amplified
(copied) a million fold. The
complementary strands of a
double-stranded molecule of DNA
are separated by heating. The
heat breaks the hydrogen bonds
holding the two DNA strands
together. When the temperature is
decreased, the primers (two small
synthetic pieces of DNA each
complementary to a specific
sequence) will bind to the
complementary sequence in the
target. DNA polymerases start at
each primer and extend the strand
by making a complementary copy
of the opposite strand. Within a
short time, exact replicas of the
target sequence have been
produced without knowing
anything about the DNA between
the primers. In subsequent cycles
(heating/cooling), double-stranded
molecules of both the original
DNA and the copies are
separated; primers bind again to
complementary sequences and
the polymerase replicate. At the
end of many cycles, the DNA pool
is greatly enriched in the small
pieces of DNA that contain the
target sequences. This amplified
genetic information is then
Figure 1
available for further analysis.
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Equipment Used in this Class
Micropipette
A micropipette is an expensive and delicate piece of equipment used to measure
very small amounts of liquid. There are several different sizes of micropipette. The
range of the pipette is always printed on the top end. Regardless of the size, they all
work in the same way. Watch the http://youtu.be/4AZHoi3hdWM to familiarize yourself
with the use of a micropipette.
Name Low Volume High Volume
Small P-20 0.5µL 20µL
Medium P-200 20µL 200µL
Large P-1000 200µL 1000µL
Setting the micropipette - On your micropipette you will see a window with numbers in it.
This tells you how many microliters the micropipette will withdraw from your liquid. The
black wheel changes the setting. Always make sure that you do not set the
micropipette any higher or lower than the range of the micropipette shown above. If you
do, it may jam or break.
Putting on a tip - You always need to use a tip when you are using the micropipette.
You should use a new tip each time you change solutions (to avoid contamination).
Hold the box of tips on the desk (so it doesn't flip over) and slide the micropipette into a
tip. Withdraw micropipette and tip from the box.
Use of the plunger - First, look at the plunger part of the micropipette. When you push
the plunger down there are two different stops. The first stop is for drawing up liquid. If
you keep pushing down you will notice that it gets a bit harder to push and you come to
the second stop. This second stop is for pushing out the liquid.
Using the micropipette - Push the plunger down to the first stop and put the tip into the
liquid. You don't need to stick the tip very far into the liquid. Release the plunger while
the tip is in the liquid. Release it slowly and smoothly so it doesn't just pop up. Take
the tip out of the liquid and push the plunger down all the way to the second stop to
push out all the liquid into your new tube. Remove the tip by pushing down on the
ejector button by your thumb and place the used tip in the trash.
NOTE: You can review the proper use of the micropipette http://bit.ly/qJzfEb.
Thermocycler
The PCR machine or thermocycler is a heating and cooling block. The block heats the
samples to 94C to make the DNA single stranded, then cools to 59C to allow primers to
bind to DNA and then finally to 72C so the polymerase will be able to make the
complementary DNA strand using the primers as starting points. We will run the
thermocycler for 40 rounds of heating and cooling, at that point there should be
sufficient DNA to observe in next week’s lab. The process takes about 4-5 hrs. so your
TA will put the finished PCR tubes in the freezer until next lab.
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PCR, Lab Session 7 - Day of Class
Experiment
PCR utilizes enzymes needed for DNA replication to make millions of copies of specific
fragments of DNA. To PCR amplify a fragment of DNA you need to know a little of the
sequence of the DNA you are interested in amplifying. Two small (20-25 bp (bp=base
paris) single strand DNA strands called primers were purchased which are
complementary to the two ends of the BT toxin DNA sequence; this is all that is needed.
For BT, one primer is 5’-ACCATCAACAGCCGCTACAACGACC-3’ (the other is not
listed but is required). The size of the resulting double-stranded PCR product is 225 bp
in length.
As a positive control, we will be using another plant gene called PSII. This gene is
found in all plants since it is a major constituent of photosystem II and is needed for
photosynthesis.
The question you will be trying to answer today and during next week’s lab is:
“Do any of the Tortilla chip brands contain transgene DNA (GMO)”?
Even after storage, grinding, harsh (lye, strong base) treatments to remove the corn
kernel cover, pressing into tortillas and frying of chips, there are still DNA fragments
large enough to be detected.
Your table will be given the choice of four bags of corn chips labeled A, B, C or D. PICK
two bags to check for the presence of GMO. You will only need one chip from each
bag. Make sure you mark down and identify (keep good notes) which bag you took the
chip from.
1. Measure out 1 gram of chip from one bag (a chip weighs about 2 grams). Break off
pieces until you get the correct weight. Place the pieces in a mortar. Repeat the
same procedure for the second bag and the GMO free corn meal sample.
2. Grind the chip/corn meal up with the pestle until it forms a fine powder.
3. Add 5 ml of distilled water to the mortar. Grind powder and water with a pestle for at
least 2 min to form the chip slurry.
4. Add 5 ml of water again and grind further until smooth enough to pipet.
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5. Prepare three tubes with an appropriate label corresponding to your samples. The
label should include your initials (or group number), day and chip bag.
6. Pipette 50ul of each ground slurry to the small plastic tubes containing 500 l of
InstaGene, Recap tube (check with the instructor to make sure you use the correct
size tube). Note: the tips used to transfer the 50 ul are very small and easily clogged
with particles of corn chips. Look at the fluid in the tip to make sure fluid comes to
the 50 ul line.
7. Shake or flick the tubes and place in a 95 C water bath/heat block for 5 minutes. The
InstaGene mixture contains a resin that removes the Calcium, Magnesium and other
ions from the solution (like a water softener) and the heat helps to liberate the DNA.
8. Place the tubes in a centrifuge and centrifuge for 5 minutes at the set speed. This
will pellet the resin and any large fragments of chips.
The supernatant now contains the DNA. Be careful not to disrupt the pellet which
will contaminate your DNA!
1. You could have contaminated your sample with GMO DNA from another sample.
2. You might not have properly heated or spun your DNA preparation, thus no DNA
in your solution.
3. You could have made good DNA but left out some component of the PCR
reaction, thus no product.
4. Or there could be a technical problem with the PCR machine, thus no product.
Here is where experimental design becomes important. The experiment you design
should take into account that you have a maximum of 7 tubes to set up (this is due to
the limitations in next week’s lab). Some tubes will be used to test for the unknown
chip DNA samples but others should be used as controls to make sure the various parts
of the PCR are working properly.
A. You have available in the lab two sets of primers; one for the BT transgene and
the other for PSII (photosystem II used in photosynthesis).
B. You have your unknown DNAs.
C. You have your DNA from a known GMO free source (corn meal).
D. You have available DNA from a known GMO source.
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1. State the hypothesis that you will be testing.
2. Using your isolated DNA samples and the four primers (A, B, C, D), design an
experiment to test your hypothesis while controlling for problems 1-4 above. This
design, when done correctly, will tell you what to put into each of the 7 PCR
tubes. Use the table below to help in your design process.
Your TA will pull you all together and discuss the experimental design you derived. Be
prepared to share with the class what you decided. After class consensus is reached
for each of the seven tubes, you will set up your 7 tubes for the PCR test. For your lab
report you will be asked to indicate why PCR was done on each of these samples so
keep good notes.
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Part E – PCR Set Up, 30 minutes (02:00)
1. It is now time to pipette samples and reagents into each of the 7 tubes. Following
the experimental design above, place 20 ul of the correct DNA into each tube. USE
a NEW tip each time! Be careful not to disturb the pellet when you take the
DNA solution. Only put your tip into the tube far enough to get the fluid, not
the resin pellet.
2. With new pipet tips, add 20 ul of the appropriate primer mix to each tube. The
primer mix contains the forward primer, reverse primer, deoxynucleotides (dATP,
dCTP, dGTP, dTTP) and the heat stable DNA polymerase enzymes needed for
PCR.
3. When all tubes are completed, cap each tube and mix each of the tubes by flicking
with your finger, then tap the bottom on the table to get the fluid to the bottom of
each tube.
4. Give your tubes to the TA who will help you load the tubes into the PCR machine.
Your TA will pull the class back together to discuss the following questions.
1. Before leaving the lab make sure you table is clean. You can discard the
remainder of the DNA samples.
2. Throw away tips and any other garbage in the regular trash.
3. Scrub the mortars and pestles with soap and water, rinse with water and leave
upside down to dry for the next class.