Instructor’s Advance Preparation Guide
Lesson 1: Synthesis of Adenine from the Chemical Inventory.
Preparation Overview:
Required Materials for 8 lab stations:
Quantity
Formamida
Ammonium Formate
Diaminomaleonitrile
Capillary tube/toothpick
Test tube
50 mL beaker
Access to an oven or sand bath
Thermometer
Spatula
Weigh boat
Test tube tongs
Test tube rack
Distilled water
10mL graduated cylinder or a transfer pipette
TLC plate
TLC developing chamber
Ruler
Pencil
Short wave UV lamp
48 ml
8.0 g
0.8 g
32
8
8
Common workstation:
Quantity
UV light
Hair dryer
Access to an oven or sand bath
Balance
Short wave UV lamp
Distilled water
8
8
8
8
8
8
8
8
8
8
1
1
2 or 3
3 or 4
1
TLC plates should be cut with the dimensions 3” X 1”
Preheat your oven to 180C or your sand bath to 240C
Teacher’s Note: A sand bath can be made from any type of sand. You can purchase
the sand from a craft store or home improvement store, or obtain it from a
playground. The size of the sand bath will be determined by the number of students
using it. A 500mL beaker sand bath can accommodate at least 5 test tubes. The
actual volume of sand used is not important; the level of the sand bath should
completely cover the sample. A sand bath to accommodate an entire class can also
be created using a large hot plate and a Pyrex dish or deep aluminum pan.
Student workstation:
Quantity
Formamide
Ammonium formate
Diaminomaleonitrile (DAMN)
Capillary tube/toothpick
Test tube
50 mL beaker
Thermometer
Spatula
Weigh boat
Test tube tongs
Test tube rack
10mL graduated cylinder or a transfer pipette
TLC plate
TLC developing chamber
Ruler
Pencil
6ml
1.0 g
0.1 g
2
1
1
1
1
3
1
1
1
1
1
1
1
Common workstation:
Quantity
UV light
Hair dryer
Access to an oven or sand bath
Balance
Short wave UV lamp
Distilled water
1
1
2 or3
3 or 4
1
Instructor’s Advance Preparation Guide
Lesson-2 Thin Layer Chromatography
Preparation Overview:
These solutions can be made by the instructor ahead of time such as a week or 1 or 2 days
before.
Standard Adenine solution- 15 min
Standard Thymine solution-15 min
Standard cytosine-15 min
Unknown-1 (Any standard solutions)
Unknown-2 (any other standard solution)
Unknown-3 (a mixture of Adenine and Thymine) - 15 min
Unknown-4 (a mixture of Thymine and cytosine)-15 min
Required Materials for 8 lab stations:
Quantity
TLC Plates
24 plates
Developing Chambers (beaker, plastic wrap, rubber band)
24 Chambers
Ruler
8
Pencil
8
Microcapillary tubes-56 tubes (each micro capillary is broken down into two pieces and
labeled to avoid the contamination.)
56 tubes
UV lamp
Tweezers
8
Vials containing prepared solutions of: Adenine, Cytosine and Thymine8 each
Unknown solution 1
8 vials
Unknown Solution 2
8 vials
Unknown Solution 3
8 vials
Unknown Solution 4
8 vials
Distilled Water
Prepare reagents:
Prepare TLC Plates - cut to the dimensions of 2”X3” (exact measurement is not
important)
Prepare Developing Chambers (beaker, plastic wrap, rubber band) - 100 mL beakers
work well. The beaker size just needs to accommodate the width and height of the plate.
Clear plastic cups would work as well.
To make capillary tubes:
You can use toothpicks instead of capillary tubes. However, students often make too
large of a spot with the toothpicks. Flat toothpicks are preferable to the thicker, rounded
ones. One long capillary tube can be separated into two smaller pointed tubes, by using
the heat from a Bunsen burner or the heat or candle. To accomplish this, hold the
opposite ends of the capillary tube; place the center over a flame when the glass starts to
soften, in one quick motion, pull capillary tube apart. Gently tap the melted end onto a
hard surface to remove any oddly shaped, bent or closed points.
To prepare the Stock solutions:
The stock solutions of Adenine, Thymine and Cytosine can be easily prepared. See the
materials sheet for ordering instructions. Guanine is not used as a nitrogenous base in this
lab series because it is not soluble in water and requires a different mobile phase in the
chromatography chamber.
Each group will use a very small amount of each of the nitrogenous base stock solutions,
so a few mL of each one can accommodate many students. They are also very stable, and
can be reused from one year to the next.
Refrigeration or freezing for long periods of storage is preferable, however.
To prepare adenine, thymine and cytosine, add 0.1 grams of base to approximately
100mL of water. If this is not sufficient to dissolve the entire base, add water 20 mL at
time, until the white solute has completely dissolved.
The unknown solutions are made from different combinations of the stock solutions. You
can use all combinations of the bases, alone, in pairs or all together. Cytosine and
thymine run fairly close together on the plate. Using a longer plate helps to separate the
solutions, but takes longer. Putting the C and T together is a good challenge to help the
students recognize the spotting.
Preparing the TLC plate.
In this step, you will be preparing your plates to be used in the chromatography chamber.
1. Using a ruler and a pencil, draw a across
the TLC plate 2.0 cm from the bottom, as
indicated in the picture below pressing the
pencil lightly as to not damage the
coating on the TLC plate. This line will
serve as the origin line. Plate 1 will be used
Origin
for determining the Rf value of the stock
Line
solution, so you will need to label the
location for the 3 different nitrogenous
bases. Again, taking care to press gently
down on the TLC plate, add 3 hash marks.
Evenly space out the marks along the plate,
starting and ending no less than 0.5 cm from
the edge of the plate. Using the letters A, C and T, label the marks under the
origin line.
2. The second plate will be used to run your 4 unknown solutions. Draw an origin
line on your second plate, identical to the first, except this time you will need to
make room for 4 hash marks. Under the origin line, label the hash marks so that it
correspond with the unknowns you are using.
Step Three: Spotting the TLC plate
You will be using capillary tubes to spot both the known and unknown solutions to your
TLC plates. Each solution will require a separate capillary tube to prevent crosscontamination. Using a piece of tape wrapped around the top of the tube, carefully
identify each tube, using the same labels you used to label the plates. Use caution with
the capillary tubes, as they are fragile and very sharp when broken.
1. Take a capillary tube labeled A and place the sharpened end into stock solution A. You
should be able to see the solution rise up into the tube, through capillary action.
2. Next, using plate #1, touch the end of the capillary tube gently to on the origin line at
the spot indicated for that solution. You do not want to scratch the plate with your
capillary tube. Your goal is to make a small spot. DO NOT let all of the contents of the
capillary tub run onto the paper. You will not use all of the solution inside the
capillary tube.
3. You will now repeat this process for the remaining solutions on plate 1, using a
different capillary tube to spot each remaining nitrogenous base at the indicated location.
4. Once you have finished spotting plate one, repeat the process for plate two, using a
different capillary tube for each unknown solution.
5. Use your tweezers to pick the plates up by the corner, and gently blow on them until
each spot is dry. When all spots on both plates have completely dried, you may proceed
to the next step.
Step Four: Developing the TLC plate
Before placing your TLC plates into its developing chamber, measure and compare the
height of the water in relation to the line you have drawn on your TLC plate. If it appears
that the water level of the chamber will be above your origin line when you place your
plate into the chamber, remove some of the water from the beaker. This step is critical! If
the water covers the line when you place the TLC plate into the developing
chamber, you will have to start the lab over from the beginning. If you are unsure if
the water will cover the line, then err on the side of caution and remove some of the
water. You can always add water back with no negative effects if there isn’t enough
water in the beaker to develop the plate.
1. Without touching it directly with your fingers, use the tweezers to carefully place the
prepared TLC plate in the developing beaker, so that it is sitting on the bottom of the
beaker, and leaning against the side of the beaker that is not covered by the filter
paper. Be very careful! If the plate falls into the water, you will have to start the lab
over from the beginning! Make sure you record what time you first placed the plate
into the chamber.
2. Cover the beaker with the saran wrap, and carefully secure the saran wrap with a
rubber band, making sure not to dislodge the TLC plate within the chamber. If your
plate falls over, you can use your tweezers to put it back into position, provided that it
did not fall over. DO NOT pick up the chamber once your plate is in place and
running.
3. Watch as the mobile phase runs up the TLC plate. When the mobile phases
approximately 1 cm from the top of the TLC plate, removes it from the chamber
using the tweezers, and places it on a paper towel. DO NOT allow the mobile phase
to overrun the top of your plate.
4. Using a pencil, trace the location of the mobile phase on your plate. This is a critical
step for the calculation of the Rf.
5. Place the first plate on a paper towel, and repeat steps 1-4 for the second plate.
6. Once both of your plates have been removed form the chamber, and the location of
the mobile phase has been indicated, hold your TLC plate with your tweezers; use a
blow dryer to completely dry your plate.
Step Five – Visualizing the spots
Warning: UV light is damaging both to your eyes and to your skin! Make sure you are
wearing your goggles and do not look directly into the lamp. Protect your skin by
wearing gloves.
1. Place plate one underneath a downward facing shortwave UV lamp. Mark all
spots that you see, no matter how faint, with a pencil, tracing around their outline.
Ideally, you should see individual dots for each compound in the mixture.
2. Repeat this step for the second plate.
3. Now that you can visualize the molecules that are on the plate, you can determine
the Rf factors for your spots, and identify the composition of the unknown
solutions.
Step Six – Evaluating the data
Plate #1 - The purpose of plate one is to determine the Rf values of your stock
nitrogenous bases.
Plate #2 – The purpose of plate two is to determine the Rf values of the unknown
solutions, so that you can compare them to the Rf factors from plate 1, and be able to
determine the composition of the solution.
Student workstation:
TLC Plates
Developing Chambers (beaker, plastic wrap, rubber band)
Ruler
Pencil
7 Micro capillary tubes
Tweezers
Vials containing prepared solutions of: Adenine, Cytosine and Thymine
Unknown solution 1
Unknown Solution 2
Unknown Solution 3
Unknown Solution 4
Distilled Water
Common workstation:
UV light
Hair dryer
Quantity
3
3
1
1
7
1
1 each
1
1
1
1
Quantity
1
1
Instructor’s Advance Preparation Guide
Lesson-3 Strawberry DNA Extraction Lab
Preparation Overview:
 Create extraction buffer for on-level classes-15 min
 Chill ethanol by putting it into an ice water bath or freezer at least 30 min. prior to the
beginning of class.-30 min to 45 min.
Required materials for 8 Lab stations
Straw berries
Ziploc Bags
For DNA Extraction Buffer
Water
Soap
NaCl
250 or 100 ml Beakers
100 ml Beakers
Cheese Cloth or coffee filters or Kim wipes
Test Tubes
Test Tube Stopper/ Papafilm/ aluminum foil
10 ml graduated cylinders
Ice Cold ethanol
Stirring Rods
Quantity
8 to 10
8
500ml
5ml
5g
8
8
Box
16
8
8
400ml
8
DNA Extraction buffer preparation:
The DNA extraction buffer is made from water, soap and NaCl. It is preferable to use a
clear soap that is free of aloe, fragrances, lotions, etc. For advanced classes, it is
recommended that students make their own buffer solution.
As a general guideline, for one class use 500 mL of water, 5 mL of soap, and 5 g of NaCl.
The amounts needed to make the buffer solution may vary and exact measurements are
not critical. This 100:1:1 ratio can be used to make larger or smaller volumes of buffer
solutions as necessary.
Note: You do not want the extraction buffer solution to be very sudsy. The buffer will
also keep for long periods of time.
Student workstation:
Quantity
Strawberry
1
Ziploc bag
1
DNA extraction buffer (made from lab soap, water and salt, instructions below)
250 mL beaker (a 100 mL beaker is also acceptable)
1
100 mL beaker
1
Cheese cloth (coffee filters/ Kim wipes also work equally well)
5
Test tubes
2
Test tube stopper/ Para film/ Aluminum foil
1
10 mL graduated cylinder
1
50 mL of ice-cold ethanol
50 ml
Stirring rod or transfer pipette
1ml
Instructor’s Advance Preparation Guide
Lesson 4: DNA Hydrolysis Lab
Preparation Overview:
3 M Hydrochloric acid
Breaking a capillary tube into 2 smaller tubes
Flag the smaller capillary tubes
Adenine standard
Cytosine standard
Thymine standard
20 min
10 min
15 min
can be used from the previous lab
Required Materials for 8 lab stations:
DNA solution as prepared in DNA Extraction Lab
Test tube
Pair of test tube tongs
Test tube rack
Rubber stopper
TLC plate
Capillary tubes/toothpicks
Ruler
Pencil
Pair of forceps
TLC chamber
10 mL graduated cylinder (or 3 transfer pipettes)
3 Molar HCL
H2O
Common workstation:
Access to a 95C - 100C hot water bath
Access to a hair dryer
Access to a UV lamp
Quantity
8
8
8
8
8
32
8
8
8
8
8
32 ml
16 ml
Quantity
1 or2
1or 2
1
Pre-lab material preparation instructions:

One capillary tube can be separated into two smaller pointed tubes, which is
suggested for optimal spotting, by using the heat from a Bunsen burner or the heat
from a candle. To accomplish this, hold the opposite ends of the capillary tube,
while placing the center over a flame. When the glass starts to soften, pull
capillary tube apart. If the pointed end of the new, smaller capillary tube is closed,






it can be gently tapped on a solid surface to break it. Advanced classes could be
allowed to complete this step on their own.
You can also use toothpicks as an alternative for capillary tubes. Flat toothpicks
are preferable to round toothpicks. Students should use the more pointed end of
the toothpick, and take care to make a small spot.
2-3 hair dryers should be sufficient for the lab. Some students may already have
hair dryers at school because of early sports practices, and may be willing to bring
them in to share with the rest of the class.
Standards of Adenine, Thymine and Cytosine can be used from the previous lab. (
2-Lab)
To make sure that there is sufficient time to heat the samples, it is recommended
that the hot water bath be turned on and brought up to temperature of 95 C
before the students come into the classroom. To create a water bath, add at least
350 mL of water to a 500 mL beaker. If the level of the water bath drops too low,
the water added to maintain the volume should be pre-heated. A significant drop
in the temperature may negatively impact the results
Approximately 60 mL of 3M HCl is needed for a 30 student class, with students
working in pairs. To prepare 60 mL of 3M HCl from a 12M stock solution, add
15 mL of 12M HCl to 45 mL of H2O. When diluting an acid, always add acid to
water, not water to acid.
Teachers can take care of removing the test tubes from the water bath after adding
acid and water to the heated sample, to if you have a regular schedule of 45 or 50
min schedule. For Block schedule it works fine.
Student Work Station
DNA solution as prepared in DNA Extraction Lab
Test tube
Pair of test tube tongs
Test tube rack
Rubber stopper
TLC plate
Capillary tubes/toothpicks
Ruler
Pencil
Pair of forceps
TLC chamber
10 mL graduated cylinder (or 3 transfer pipettes)
3 Molar HCL
H2O
Quantity
1
1
1
1
1
4
1
1
1
1
1
4 ml
2ml
Common workstation:
Access to a 95C - 100C hot water bath
Access to a hair dryer
Access to a UV lamp
Quantity
1 or2
1or 2
1
Instructor’s Manual: Post Lab Activity:
Lab 1: Synthesis of Adenine Lab
Answers to Post-Lab Questions:
1. Why did the densest gases sink to the center of the enormous, hot cloud that
was primordial Earth instead of sinking to the bottom of the cloud? (Hint:
Refer to the background section.)
The densest gases sank to the center of primordial Earth because the circular
cloud that was Earth rotates and the “bottom” of the circle at one point can easily
become the “top”. The densest material must sink only to the center, as is that is
the only place within a circle that does not move during its rotation
2. List the four nitrogenous bases that are found in DNA. Make sure to
indicate which bases are purines and which are pyrimidines.
Purines: adenine and guanine. Pyrimidines: thymine and cytosine.
3. Did you think it would be this easy to create an essential component of life?
Write down your thoughts on the significance of creating a nitrogenous base
in the classroom.
Any answer is acceptable.
3. Which nitrogenous base did you hypothesize would be generated? Did your
hypothesis match your result?
Answers will vary.
Additional Questions (for Chemistry classes):
1. Identity and draw at least one functional group found in a molecule from the
background.
Answers may vary
2. Identify the number of sigma and pi bonds in the formamide structure
(refer to background section).
3 sigma bonds, 1 pi bond (make sure they identify these bonds on the formamide
structure located in the background section)
3. How do you think HCN was formed?
HCN was formed through atmospheric interactions among early Earth substances.
4. How many lone pairs of electrons are on the nitrogen atom in the DAMN
molecule?
1 lone pair
Lab 2: TLC Chromatography Lab
Answers for the Post Lab Questions: TLC Chromatography Lab.
Answers may vary depending on the unknown.
Lab 3: DNA Extraction Lab
Answers for the Post Lab questions.
1. Commonly cultivated bananas are usually triploid and wheat is usually
hexaploid. How many of each type of chromosome do you think they have?
Triploid organisms contain 3 copies of each type of chromosome, and hexaploid
contain 6.
2. In some species of bees, wasps and ants, males develop from an unfertilized
egg, and are monoploid as a result. How many of each type of chromosome
do you think they have? What is a more common name for monoploid?
They have 1 copy of each type off chromosome, which is also called haploid.
3. In step 9, you are instructed to pour your ethanol down the side of the
beaker. Why do you think that is an important step?
To prevent the ethanol and the water from mixing, creating two separate layers
that cause DNA to precipitate at the top.
4. In the background section, 4 major steps were listed. Identify each step, and
determine which steps in the protocol they match with.
Step
1
2
3
4
Purpose
Break apart the cell wall
Distrupt the cell and nuclear membranes to
release the DNA
Filter DNA from excess strawberry material
Precipitate DNA
Related Procedure Step(s)
1,2
3
4,5,6
8,9
5. Construct a concept map using at least 10 of the bold words found in the
background.
Student answers may vary. Number of words used can be increased for advanced classes
or used as an extra credit opportunity.
Lab 4: DNA Hydrolysis Lab
Answers for the Post lab Questions.
Post-Lab Questions:
1. The definition of chemical hydrolysis is given, what do you think enzymatic
hydrolysis means?
Enzymatic hydrolysis is the breaking down of DNA using enzymes. (E.g. D Nase)
2. Is DNA soluble in water? How do you know?
DNA is soluble in water, because of the “like dissolves like” principle and both
substances are polar. Also, they observed this property in the DNA Extraction Lab.
3. Draw a picture that illustrates the term ‘anti-parallel’.
Any picture illustrating anti-parallel strands of DNA is acceptable
4. Which of DNA’s nitrogenous bases are purines and which are pyrimidines?
Purines: adenine and guanine. Pyrimidines: thymine and cytosine
5. Construct a paragraph using at least 10 of the bolded words.
Student answers will vary
Additional Questions (for Chemistry classes):
6. Give three examples of strong acid.
HCl, HI, HNO3, H2SO4, HClO4, etc
7. Describe how the arrangement of electrons leads to the formation of partial
charges?
The more electronegative element attracts the electrons towards its side, which causes
electrons to move closer to that atom, giving it a partially negative charge. Therefore, this
causes the other atom in the bond becomes partially positive.
8. Explain the difference of the attraction between partial charges verses the
attraction of ions.
The attraction between partial charges is a temporary and weak. However, the attraction
between ions is very strong because this is between permanently negatively and
positively charged.