Appendix A:
Instructor’s Advance Preparation Guide & Protocol
Lesson 1: Synthesis of Adenine from the Chemical Inventory.
Preparation Overview:
TLC plates should be cut with the dimensions 2” X 1”- 15 min
Preheat your oven to 180C or your sand bath to 240C – 20 min
The actual amounts of ammonium formate, formamide and DAMN can be changed
depending on the stock available. However, a 1g: 6mL: 0.1g ammonium formate:
formamide: DAMN is required. Accurate measurements by the students are very
important. The amount of reaction mixture produced by the quantities listed in the lab
protocol is much greater than required for student use.
Reduction of reactants is recommended if equipment capable of measuring smaller
quantities is available.
This experiment can also be done as a demo in the lab and give the reaction mixture
for students to spot it on the TLC plates. This saves the use of chemicals.
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
8
8
8
8
8
8
8
8
8
8
1
Hair dryer
Access to an oven or sand bath
Balance
Short wave UV lamp
Distilled water
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
1
1
2 or3
Balance
Short wave UV lamp
Distilled water
3 or 4
1
Background Information
Astronomers tell us that billions of years ago, the Earth existed only as an enormous, extremely
hot cloud of gas. While there is no way to measure directly the heat that must have prevailed in
the gas cloud, it was certainly considerably higher than what we can artificially obtain today. It
would have been so hot that it was not possible for even the simplest compounds to exist.
Instead, this future earth gas cloud would have consisted only of atoms.
Under the influence of the same gravitational force that attracts today’s matter towards the center
of the earth, those atoms that made up the primordial gas cloud would have also been affected by
gravity. This would have caused the gases forming the cloud to arrange themselves into layers,
with the densest gases sinking to the center and the least dense ones remaining on the outer edges
of the cloud. Over time, as the cloud cooled, the cloud’s material began to change. The atoms in
the center of the cloud formed a molten liquid, while the least dense elements remained an
envelope of gas.
Continued cooling allowed for first the formation of simple molecules, which were able to
become more complex, gradually forming a solid envelope surrounding the thick, hot center.
This envelope eventually gave rise to the crust of the earth, separating the hotter core from the
gaseous atmosphere.
As the molten center of the earth continued to cool, it contracted. The more rigid layer, unable to
follow the contraction of the liquid center, formed cracks and folds through which the red-hot
center poured out onto the surface of the earth. Modern geologic events, such as volcanic
eruptions and mountain building, provide evidence that the contraction of the earth’s core
continues today.
Primitive Earth’s gaseous atmosphere at first consisted of just those atoms found in the lightest
layers of the initial cloud of dust. Slow cooling of the atmosphere introduced chemical diversity
by the spewing of gases by the ancient volcanoes, allowing more complex molecules to form.
Among those molecules formed would have likely been formamide (H2NCOH) and hydrogen
cyanide (HCN), which may have played a large role in the chemical processes that allowed for
the formation of the first life forms.
Finally, when the earth’s temperature fell to 100C, it became possible for the water in the
atmosphere to exist in the form of liquid drops. Continuous downpours of rain fell upon the
surface of the earth, inundating it in the form of a boiling ocean. Organic molecules that were
previously formed in the atmosphere dissolved into the liquid drops and accompanied them to
the Earth’s watery surface. Floating in the water that would become the birthplace of the Earth’s
first life forms, those compounds met. Combining with each other, they formed large and
complicated molecules, including those that are required and exclusively utilized by living
things.
In this lab, you will be using materials that were available in earth’s early atmosphere to generate
and identify nitrogenous bases using TLC chromatography. The reactants used in this lab are
formamide, ammonium formate, and diaminomaleonitrile (DAMN). As explained earlier,
formamide is thought to have been readily available in early earth. This hypothesis is further
supported by the discovery of its presence in stars, comets, and other interstellar objects. The
other reactants, ammonium formate and DAMN, can be readily created from either formamide or
hydrogen cyanide.
The reaction below illustrates how hydrogen cyanide can be hydrolyzed to form formamide
(reaction A), which can then be hydrolyzed to form ammonium formate (reaction B).
Diaminomaleonitrile is formed from hydrogen cyanide according to the following reaction.
There are intermediate forms in the reaction process, but the actual mechanism is currently
unknown.
Procedure
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP IN THE LAB.
Step 1: Obtain and label a clean test tube.
Step 2: Using the weigh boat, measure 1.0 gram of ammonium formate and place it into your
test tube.
Step 3: Using a graduated cylinder, measure 6.0 mL of formamide and add it to the test tube
containing the ammonium formate.
Step 4: Stir the solution with a stir rod, and observe. Record the physical appearance of your
solution on your data sheet.
Step 5: Securely cover your test tube, and place it into the sand bath for 5 min. Record the time
on your data sheet and calculate when you need to remove your sample, and also record that time
on your data sheet. Proceed to step six while your sample is in the sand bath.
Step 6: Using your weigh boat, measure exactly 0.1 grams of the diaminomaleonitrile (DAMN)
and set aside for use in step 8.
Step 7: After 5 min., using your test tube tongs, remove your sample from the sand bath and
place it into a test tube rack. If all of the solid ammonium formate has dissolved, then move to
step 8. If solid material remains in your test tube, return your test tube to the sand bath for an
additional 3 min. before moving to step 8.
Step 8: Record the physical appearance of your solution on your data sheet. Once your test tube
has cooled to the touch, carefully add the DAMN you measured in Step 6 into your test tube and
thoroughly mix with a stirring rod.
Step 9: Securely cover your test tube. Place your thumb over the top of the covering on the test
tube and invert your test tube several times, to get all of the DAMN off of the sides of the tube.
Record the physical appearance of your solution on your data sheet.
Step 10: Place your test tube back into the sand bath for 20 min. Record the time of entry on
your data sheet and calculate when you need to remove your sample, and also record that time on
your data sheet. While your test tube in the sand bath, prepare your TLC plate and chamber. You
will need room for 4 spots on your plate, yours and samples from three other groups.
Step 11: After 20 min., using your test tube tongs, remove your sample from the sand bath, and
place it into a test tube rack to cool. Record the physical appearance of your solution on your
data sheet.
Step 12: When the test tube is cool to the touch, take your covered mixture to the fume hood.
Uncover, and measure 1 mL of the reaction mixture and place it into small beaker. Place the
remaining DAMN reaction mixture into the designated waste container inside the fume hood.
Step 13: Take your sample back to your lab bench. Add 10 mL of water to your reaction
mixture, and stir. Use this to spot your TLC plate in the location labeled for your sample. If you
have forgotten how to set up and run your TLC plate, refer to the TLC instruction sheet.
Step 14: Spot samples from 3 other groups onto your plate at the appropriate locations. Make
sure your spots are completely dry, and then run your TLC plate in the chamber, and analyze
your results using the UV lamp.
NOTE: UV light can be damaging to the skin and eyes. Avoid looking directly into the UV
light, and keep it facing down at all times.
Step 15: Calculate the Rf of your sample, and identify the unknown nitrogenous base that has
been synthesized.
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
Student Protocol
Lesson: Thin Layer Chromatography- Identification of Unknown solutions using
Chromatography.
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
Purpose:
Quantity
3
3
1
1
7
1
1 each
1
1
1
1
Quantity
1
1
To become familiar with the principles and terminology of thin layer
chromatography (TLC) to identify an unknown molecule based on comparisons with a
known laboratory standards and Rf values.
Background
Thin Layer Chromatography:
Chromatography is a method of separating and identifying mixtures of two or more
compounds. The separation is accomplished by the distribution of the mixture between
two phases: one that is stationary and one that is moving or mobile. Chromatography
works on the principle that different compounds will have different solubilities and
adsorption to the two phases, which will allow for their separation.
Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases
are a solid, stationary phase and a liquid, mobile phase. The stationary phase you will be
using in today’s lab is a plastic plate covered with an adsorbent, in this case, silica gel.
Alumina is another common solid phase used. The speed at which the molecules will
move up the plate depends on the relative difference in polarity between the stationary
and mobile phases, and will vary depending on the nature of the stationary and mobile
phases used for separation.
The following are some common uses of thin layer chromatography:
1. To determine the number of components in a mixture.
2. To determine the identity of two substances.
3. To monitor the progress of a reaction
The difference each molecule travels along the adsorbent in relation to how far the
mobile phase has traveled is called the Retention Factor (Rf) and can be used to identify
molecules, as the value is specific to each molecule, but can vary depending on the which
mobile phase and type of solid phase, or plate, that is plate that is used.
Procedure for TLC
READ ALL INSTUCTIONS BEFORE BEGINNING EACH STEP IN THE LAB
Step One: Preparation of the Developing Chamber
1. Obtain two developing chamber from the front of the room. The developing chamber
consists of a beaker, a piece a saran wrap, a piece of filter paper and a rubber band
2. Place a piece of filter paper into the beaker, as demonstrated by the image below.
This will ensure that the TLC plate will remain saturated with the vapor from the
aqueous mobile phase, so that the plate will run correctly. If the filter paper is not
already cut, then trim it so that it fits well into the beaker you will be using. It should
cover no more than ½ of the inside of the beaker.
3. Carefully pour water into the beaker, to a depth of approximately ½ cm, making sure
that the entire filter paper is saturated, swirling if necessary.
Step Two: Preparation of the TLC plate.
In this step, you will be preparing your plates to be used in the chromatography chamber.
Make sure that you press the pencil lightly when drawing on the TLC plate. You do
not want to scratch the coating on the plate.
1. On the side of the TLC plate that has the
white coating (the non-shiny side), use a
ruler and a pencil (a pen will NOT work)
to draw a line across the TLC plate 1.0 cm
from the bottom, as indicated in the picture
at left, pressing the pencil lightly so as not
Origin
to damage the coating on the TLC plate.
Line
This line will serve as the origin line. Plate
Hash mark
1 will be used for determining the Rf value
of the stock solution, so you will need to
label the location for the 3 different
nitrogenous bases. Again, taking care to
press gently down on the TCL plate, add 3
hash marks (see the picture as an example). 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 has marks so that
the 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.
7. 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.
8. 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 into the water. DO NOT pick up the chamber once your plate is in place
and running.
9. Watch as the mobile phase runs up the TLC plate. When the mobile phase is
approximately 1 cm from the top of the TLC plate, remove it from the chamber using
the tweezers, and place it on a paper towel. DO NOT allow the mobile phase to
overrun the top of your plate.
10. Using a pencil, trace the location of the mobile phase on your plate. This is a critical
step for the calculation of the Rf.
11. Place the first plate on a paper towel, and repeat steps 1-4 for the second plate.
12. 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.
4. 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.
5. Repeat this step for the second plate.
6. Now that you can visualize the molecules that are on the plate, you can determine
the Rf values 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.
Use your data sheet to calculate the Rf value for each of your stock solutions as well as
the spots you visualized in the unknown solutions. Once you determine the Rf value for
each stock nitrogenous base, copy this information into your science binder in a safe
place. You will need these values for other labs throughout the year, and if you lose it,
you will have to redo this portion of the lab.
Calculating the Rf Value
Sample Calculation: This calculation is based upon a sample that is made up of a
combination of substances, and therefore has more than one spot. The procedure for
determining the Rf value for each spot within one sample is the exact same as the
procedure for determining the Rf value for many individual samples on one plate.
The solvent front is the distance the mobile phase traveled on the plate, and is what you
recorded with your pencil when you took your plates out of the developing chambers.
Make sure you measure from the center of the spot to the origin line to get the distance
moved by the molecule.
1
The formula for calculating the Rf values is:
Rf = Distance moved by the molecule / Solvent front
The Rf value for the substance indicated by #1 would be:
Rf = 5.5 cm/6.0 cm = 0.92
Lab-3: Extraction of DNA from Strawberries Lab
Purpose: To extract the DNA from Strawberries.
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
Background Information
In today’s lab, you will break apart the cells of a strawberry and release its D
deoxyribonucleic Acid into a form that is visible to the naked eye. Wild strawberries are
diploid, meaning they contain 2 copies of each chromosome. Commercially produced
strawberries are octoploid. Because they contain 8 copies of each chromosome, they are
particularly good material to use for this lab.
The DNA in today’s lab will precipitate, or come out of solution, as long white strands.
This white material is actually thousands of DNA strands (and the associated proteins)
wrapped around each other. An individual DNA strand is so small, it can only be imaged
by the most sophisticated and specialized equipment. Remember, the images that helped
Watson and Crick to uncover the double helix structure of DNA were taken by
Rosalind Franklin using X-Ray Crystallography.
Several steps are required to process the DNA so that it will precipitate out into a visible
form. First, the cell wall must be broken open. This is accomplished, in part, by the
physical act smashing the strawberries in a Ziploc bag. Ripened fruit is also used in this
lab because the cell wall is already weakened by the ripening process.
The second step in the process requires the rupturing of the cell and nuclear membranes
to free the DNA. This is accomplished by the addition of an aqueous extraction buffer,
comprised of detergent and salt. Unlike DNA, which is formed from nucleotide
monomers made of deoxyribose, phosphate and a nitrogenous base, cell and nuclear
membranes contain primarily fats and proteins. Because the chemical nature of the
membranes and DNA is different, the extraction buffer disrupts them while leaving the
DNA in tact.
The third step is the separation of the DNA from the bulkier, excess strawberry material,
which is accomplished by filtering the DNA mixture. The final step in the process is to
precipitate the DNA. DNA is soluble in water, therefore, not visible in the filtered
strawberry mixture. However, DNA is insoluble in ethanol. Therefore, the careful
addition of ethanol to the top of the DNA mixture will cause the DNA to rise to the top
and form visible, insoluble threads that can be spooled and collected.
Procedure
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP IN THE LAB
Step 1: Remove the green stem from one strawberry, and place into a plastic freezer bag.
Step 2: Remove the air from the bag, seal it and mash the strawberry for 2 min. or until it
is completely broken apart.
Step 3: Add 25 mL of DNA extraction buffer to the bag, reseal the bag, and mash again
for 1 min. or until the solution is thoroughly mixed with strawberry.
Step 4: Hold the filter paper securely over the top of a 250 mL beaker, and slowly pour a
small amount of the strawberry mixture onto the filter paper. Make sure that the filter
paper does not fall into the beaker.
Step 5: To speed up the filtration process, gently squeeze the filter paper. Squeezing the
filter paper too hard will tear it, causing solid mixture to fall into the beaker and mix with
the liquid portion. If this occurs, the solution will need to be re-filtered.
Step 6: Repeat steps 4 and 5, using new filter paper if necessary, until all of the
strawberry mixture has been filtered
Step 7: Record the volume of the filtered strawberry solution on your data sheet
Step 8: Using a separate beaker, obtain ice-cold ethanol equal to 3X the amount of
filtered strawberry solution. Determine amount of volume you will need, and write it on
your data sheet.
Step 9: Slowly pour the ice-cold ethanol obtained in step 8 along the inside wall of the
beaker. The ethanol should form a layer on top of the filtered solution.
Step 10: You should immediately observe whitish DNA precipitate of out of solution at
the interface between the ethanol and the filtered solution. Make a sketch of what you
see, and record observations about what you see on your data sheet.
Step 11: Allow the DNA solution to sit undisturbed for 5 min. Remove the DNA that
has accumulated at the surface by spooling it with a stirring rod or using a transfer pipette
and place it into a test tube. If using a transfer pipette, remove as little of the ethanol as
possible.
Step 12: Remove any excess ethanol from your test tube by carefully using a transfer
pipette, or by using the corner of a paper towel to wick up any excess liquid. Ideally,
when you are finished, the only substance that will be in your test tube is DNA.
Step 13: Fill a 1 ml transfer pipette with water, and add half the contents to the test tube
containing the DNA. Cover the top of the test tube with your thumb, and vigorously
shake the test tube for 30 seconds.
Step 14: Continue adding water, half a pipette (0.5 mL) at a time, shaking for 30 seconds
after each addition, until the majority of the DNA has dissolved. Do not add more than
1.5 mL of water (1 ½ pipettes) to your sample without your teacher’s approval.
Note: Dissolving the DNA should cause the solution to become thicker and cloudier,
and most of the white strands of the DNA will disappear. Record the amount of water
used on your data sheet.
Step 15: Place a stopper into the test tube, identify your test tube and place into the
designated location.
Lab 4: Hydrolysis of DNA 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
Common workstation:
Quantity
1
1
1
1
1
4
1
1
1
1
1
4 ml
2ml
Quantity
Access to a 95C - 100C hot water bath
Access to a hair dryer
Access to a UV lamp
1 or2
1or 2
1
Background Information
The DNA that comprises all living things, from bacteria to humans, is always made up of
the exact same major components: deoxyribose, phosphate groups, and nitrogenous
bases. The difference in the amount of DNA and the specific arrangement of the
nitrogenous bases is what accounts for the wide variation in all of Earth’s life forms. In
very similar organisms, like humans and other primates, the DNA is almost identical.
Examining the similarity within the DNA can help scientists determine the relatedness
between two different species and between different individuals within a population.
I
In eukaryotic cells, DNA can be found in the nucleus and the mitochondria, as well as the
chloroplast in photosynthetic organisms. DNA is arranged like a twisted ladder, with the
backbone (sides of the ladder) being made up of repeating deoxyribose and phosphate
groups. Each deoxyribose is attached to one of four possible nitrogenous bases, which
make up the center (or rungs). DNA’s shape is called a double helix because the
molecule is made up of two strands. The two strands of DNA are anti-parallel, meaning
that they run in opposite orientations. Notice how the sugar molecules in the picture
below are oriented differently on the opposite sides (Figure 1).
There are four nitrogenous bases found in DNA:
adenine, thymine, cytosine, and guanine. These
bases can be categorized into two classes based on
structure: purines and pyrimidines. Purines have
two carbon-nitrogen rings, while pyrimidines only
have one (Figure 2). In DNA molecules, one
purine must pair with one pyrimidine. Thus,
adenine pairs with thymine and guanine pairs with
cytosine, and form base pairs. Hydrogen
bonding between the base pairs is what keeps the
double helix together.
Figure 1
Figure 2
In today’s lab, you will be using the DNA that you extracted in the previous lab, and
break it apart to release one of its nitrogenous bases. Since the nitrogenous bases all have
slightly different chemical structures, the bond strength which attaches them to sugar
phosphate backbone varies. The process of breaking apart a macromolecule, such as
DNA, into its monomers and smaller building blocks is called hydrolysis, and requires
the use of water.
The bonds that hold an individual water molecule together are polar covalent bonds.
Polar bonds exist because of an unequal attraction for electrons shared by neighboring
atoms within one water molecule. In water’s case, the oxygen is more electronegative, or
has a higher attraction for the shared electrons than hydrogen. This causes oxygen to have
a partial negative charge and hydrogen to have a partial positive charge. This type of
bonding makes water a polar molecule. A hydrogen bond is created by the electrostatic
attraction between a partially positive hydrogen atom attached to an electronegative
atom like oxygen or nitrogen and a partially negative oxygen or nitrogen atom in
neighboring molecules, and is what causes two adjacent water molecules to be attracted
to each other. Hydrogen bonding also is responsible for base pairing in DNA. DNA is a
polar molecule, in part because of the phosphate groups.
The second step required to break apart the DNA molecule is a strong acid, such as the
HCl used in this lab. The strength of an acid is determined by how readily it ionizes, or
breaks apart, in water. Here the acid works by protonating, or donating a hydrogen, to
the nitrogenous bases, which cleaves the base from the sugar-phosphate backbone. After
breaking apart the DNA molecule, you will use thin layer chromatography (TLC) to
determine the identity of the cleaved base.
Procedure:
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP IN THE LAB.
DNA Hydrolysis Lab: Part 1
Step 1: Obtain a 500 mL beaker and a thermometer. Fill the beaker with 400 mL of water
and place it onto the hot plate. Turn the hot plate on to medium. Your teacher may have
already completed this step for you.
Step 2: Obtain and label a new test tube with your group name/number.
Step 3: Using a graduated cylinder, measure out 2mL of your extracted DNA solution
and place it into your new test tube.
Step 4: Cover the new test tube. When your hot water bath has reached at least 95C,
place your test tube into the water bath and record the time your sample enters the water
bath on your data sheet.
Step 5: Heat the sample for 30 min, and write down any changes that you observe. Make
sure you adjust your temperature so that your water bath stays close to 95C. Record the
temperature of your hot water bath every 10 min. for 30 min.
Step 6: Using test tube tongs, remove your test tube from the water bath and place it into
a test tube rack. Make observations about your DNA solution, specifically noting color
and composition.
DNA Hydrolysis Lab: Part 2
Step 1: Obtain the DNA solution that you prepared in the first part of the lab.
Step 2: Carefully add 2 mL of water and 4 mL of 3M HCl to the DNA solution. NOTE:
HCl is a strong acid. Use caution when handling this substance, and follow all
appropriate safety precautions.
Step 3: Cover your test tube, and place it into a 95C hot water bath. Record the time.
Measure the temperature every 10 min. and record the information on your data sheet.
Step 4: Heat the sample for 30 min, and record any changes that you see.
Step 5: While your sample is heating, prepare your TLC plate and your TLC chamber.
You will be running 4 total samples on your TLC plate, yours and the samples created by
three other groups. If you do not remember how to set up either the TLC chamber or
plate, refer to the TLC instruction sheet.
Step 6: After your sample has heated for 30 min, remove it from the hot water bath using
test tube tongs.
Step 7: When the test tube is cool to the touch (2-3 min), pour the contents of your test
tube into a 25 mL beaker, and use this to spot your plate in the location designated for
your sample.
Step 8: Spot samples from 3 other groups onto your plate at the appropriate locations.
Make sure your spots are completely dry, and then run your TLC plate in the chamber.
Step 9: Allow the mobile phase to run up ¾ of the plate. When it is approximately 1 cm
from the top of the plate, remove it from the chamber and immediately trace the location
of the water line.
Step 10: Dry your plate using the hair dryer, and use the short wave portion of the UV
light to visualize the spots and trace their location on your TLC plate. NOTE: UV light
can be damaging to the skin and eyes. Avoid looking directly into the UV light, and
keep it facing down at all times.
Step 11: Calculate the Rf of your sample, and identify the unknown nitrogenous base that
has been hydrolyzed.
APPENDIX B: Student Worksheets.
Appendix B: Synthesis of Adenine Lab.
Mass of ammonium formate: __________
Volume of formamide: __________
Mass of DAMN: ___________
Temperature of oven/sand bath in C: ______
Temperature of oven/sand bath in F: ______
Step 5: Time into oven/sand bath:
Time out of oven/sand bath:
Step 10: Time into oven/sand bath:
Time out of oven/sand bath:
Step
Work Space for temperature conversions
C = (5/9)*(F-32)
F= (9/5)*(C+32)
______
______
______
______
Description of physical appearance
4
8
9
11
Sketch the TLC plates, before and after development.
Before
Substance
Spot 1
Spot 2
Spot 3
After
Calculations
Rf Value
Spot 4
Average
IDENTITY OF UNKNOWN NITROGENOUS BASE: _____________________
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.)
2. List the four nitrogenous bases that are found in DNA. Make sure to indicate
which bases are purines and which are pyrimidines.
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.
4. Which nitrogenous base did you hypothesize would be generated? Did your
hypothesis match your result?
Additional Questions (for Chemistry classes):
5. Identify and draw at least one functional group found in a molecule from the
background.
6. Identify the number of sigma and pi bonds in the formamide structure (refer to
background section).
7. How do you think HCN was formed?
8. How many lone pairs of electrons are on the nitrogen atom in the DAMN
molecule?
Lab: 2
Appendix B: Identify the Unknowns using the data collected:
Name: _________________________________________
Date: __________
Student Work Sheet
Sample
Distance
Traveled
Solvent
Front
Rf Value
Adenine Standard
Thymine Standard
Cytosine Standard
Unknown 1
Unknown 2
Unknown 3
Unknown 4
Provide a sketch of your TLC Plates below, making sure to include the Rf Values on the
sketch.
Student Solution Sheet:
What is your prediction for the chemical identity of?
Unknown 1:__________________________________________
Unknown 2:__________________________________________
Unknown 3:__________________________________________
Unknown 4;__________________________________________
Justify your answer for each unknown using as much evidence as you can. Evidence may
be either qualitative or quantitative in nature. Make sure you use the correct terminology
when referring to the components of the experiment!! Use your justification to convince
me that you understand how the TLC process works.
Additional Post Lab Questions:
3. On a scale from 1 – 5, how confident are you that your unknown’s were identified
correctly (5 being most confident)
4. What applications might this process have in other areas of science?
5. What were potential sources for error in this experiment?
Appendix B- Lab: 3 Extraction of DNA from Strawberries Lab
Student Data Sheet – DNA Extraction and Identification
Objective of DNA Extraction Lab: _________________________________________
_______________________________________________________________________
_______________________________________________________________________


Volume of filtered strawberry solution: __________
Volume of ethanol necessary (3X the amount of strawberry solution): _________
Sketch of DNA solution/precipitate:
Observations about DNA Solution:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Volume of H2O used to dissolve the DNA: ____________
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?
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?
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?
4. In the background section, 4 major steps were listed. Identify each step, and
determine which steps in the protocol they match with.
Step
Purpose
Related Procedure Step(s)
1
2
3
4
9. Construct a concept map using at least 10 of the bold words found in the background.
Lab: 4
Appendix B: Hydrolysis of DNA from Strawberries Lab.
Name: _________________________________________
Date: __________
Student Data Sheet - DNA Hydrolysis Lab
Objective of DNA Hydrolysis Lab: _________________________________________
________________________________________________________________________
________________________________________________________________________
Step 1:
Time in water bath: _____________
Time out water bath: ____________
Temperature of water bath:
0 min: ___________
10 min: ___________
20 min: ___________
30 min: ___________
Step2:
Time in water bath: _____________
Time out water bath: ____________
Temperature if water bath:
0 min: ______
10 min: ______
20 min: ______
30 min: ______
Observations of DNA solution Step 1:
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_
Observations of DNA solution Step 2:
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
Sketch your TLC plates, before and after development.
List the Rf values for:
Adenine: _______
Thymine: _______
Cytosine: _______
Before
After
Calculate the Rf values. Show your work for credit, and use the TLC information sheet
if necessary.
Substance
Rf Value
Calculations
Spot 1
Spot 2
Spot 3
Spot 4
Average
IDENTITY OF UNKNOWN NITROGENOUS BASE: ___________________
Post-Lab Questions:
1. The definition of chemical hydrolysis is given. What do you think enzymatic
hydrolysis means?
2. Is DNA soluble in water? How do you know?
3. Draw a picture that illustrates the term ‘anti-parallel’.
4. Which of DNA’s nitrogenous bases are purines and which are pyrimidines?
5. Construct a paragraph using at least 10 of the bolded words.
Additional Questions (for Chemistry classes):
6. Give three examples of strong acids.
7. Describe how the arrangement of electrons leads to the formation of partial
charges.
8. Explain the difference between the attraction of partial charges to one another and
the attraction of ions.
Appendix C: Glossary
Chromatography: A physical separation method in which the components of a mixture
are separated by differences in their distribution between two phases, one of which is
stationary (stationary phase) while the other (mobile phase) moves through it in a definite
direction.
Adsorbent: to gather (a gas, liquid, or dissolved substance) on a surface in a condensed
layer:
Mixture: an aggregate of two or more substances that are not chemically united and that
exist in no fixed proportion to each other.
Retention Factor: describes the ratio of time spent in the stationary phase relative to
time spent in the mobile phase.
Adenine: A purine derivative. It is one of the major component bases of nucleotides and
the nucleic acids DNA and RNA.
Cytosine: A pyrimidine base, C4H5N3O, that is the constituent of DNA and RNA
involved in base pairing with guanine.
Thymine: A pyrimidine base, C5H6N2O2, that is an essential constituent of DNA.
Solvent front: In paper chromatography, the wet moving edge of the solvent that
progresses along the surface where the separation of the mixture is occurring.
TLC: Thin Layer Chromatography
Formamide: Formamide: It is also known as methanamide, is an amide derived from
formic acid. It is a clear liquid which is miscible with water and has an ammonia-like
odor.
Ammonium Formate: ( NH4HCO2) It is the ammonium salt of formic acid. It is a
colorless, hygroscopic, crystalline solid.
Diaminomaleonitrile: It is an organic chemical that contains two amine groups and two
nitrile groups bound to an ethylene backbone.
Purines: It is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring
fused to an imidazole ring.
Pyrimidine: It is a heterocyclic aromatic organic compound similar to benzene and
pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring.[1] It
is isomeric with two other forms of diazine. Three nucleobases found in nucleic acids,
cytosine (C), thymine (T), and uracil (U), are pyrimidine derivatives:
Octoploid: having 8 sets of chromosomes.
Deoxyribose nuclei acid: (DNA) is a nucleic acid that contains the genetic instructions
used in the development and functioning of all known living organisms and some viruses
Precipitate: is the formation of a solid in a solution or inside another solid during a
chemical reaction or by diffusion in a solid.
X-ray Crystallography: It is a method of determining the arrangement of atoms within a
crystal, in which a beam of X-rays strikes a crystal and diffracts into many specific
directions
Nucleotide: It is a molecule that, when joined together, make up the structural units of
RNA and DNA. In addition, nucleotides play central roles in metabolism.
Monomer: It is an atom or a small molecule that may bind chemically to other
monomers to form a polymer or cluster.[1] The most common natural monomer is
glucose,
Solubility: It is the property of a solid, liquid, or gaseous chemical substance called
solute to dissolve in a liquid solvent to form a homogeneous solution of the solute in the
solvent.
Eukaryotic cell: A single-celled or multicellular organism whose cells contain a distinct
membrane-bound nucleus.
Hydrogen Bonding: Interaction involving a hydrogen atom located between a pair of
other atoms having a high affinity for electrons; such a bond is weaker than an ionic bond
or covalent bond but stronger than van der Waals forces.
Hydrolysis: Decomposition of a chemical compound by reaction with water, such as the
dissociation of a dissolved salt or the catalytic conversion of starch to glucose.
Polar Molecule: A molecule possessing a permanent electric dipole moment due to
unequal sharing of electrons.
Non-Polar molecule: It is a molecule that shares electrons equally and does not have
oppositely charged ends.
Protonation: is the addition of a proton (H+) to an atom, molecule, or ion. Protonation
is the reverse of deprotonation
Ionizes: To Convert into ions.
Electrostatic force: Force on a charged particle due to an electrostatic field, equal to the
electric field vector times the charge of the particle.
Partial Charge: Partial charges are created due to the asymmetric distribution of
electrons in chemical bonds.
Appendix D: Teacher Answer Guide.
.
Pre-Lab Questions:
1. What is the name of the technique you will be using in today’s lab?
a. Thin Layer Chromatography
b. Mass Spectrometry
c. Thick Liquid Calorimetry
d. Liquid Chamber Chromatography
2. How many plates will you be using in today’s lab?
a. 1
b. 2
c. 3
d. 4
3. What is the purpose of the lab?
a. To measure the rate of the movement of molecules in a solution
b. To identify the composition of unknown substances
c. To determine the speed of a chemical reaction
d. To isolate and separate different DNA nucleotides
4. Which of the following is a piece of equipment you will be using in today’s lab?
a. A Bunsen Burner
b. A calorimeter
c. A UV Lamp
d. A triple beam balance
5. TLC plates separate molecules on the basis of:
a. Polarity
b. Color
c. Size
d. Bond orientation
Lab: 1-Answers for the Synthesis of Adenine Lab 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.
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
Diaminomalenonitrile molecule?
1 lone pair
Lab: 2- Answers for the Post Lab Questions: TLC Chromatography Lab.
Answers may vary depending on the unknown.
Lab: 3-Answers for the DNA Extraction Lab
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 to 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-Answers for the DNA Hydrolysis Lab
Post-Lab Questions:
9. 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. DNase)
10. 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.
11. Draw a picture that illustrates the term ‘anti-parallel’.
Any picture illustrating anti-parallel strands of DNA is acceptable
12. Which of DNA’s nitrogenous bases are purines and which are pyrimidines?
Purines: adenine and guanine. Pyrimidines: thymine and cytosine
13. Construct a paragraph using at least 10 of the bolded words.
Student answers will vary
Additional Questions (for Chemistry classes):
14. Give three examples of strong acid.
HCl, HI, HNO3, H2SO4, HClO4, etc
15. 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.
16. 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.
Appendix E: References
Hill, A.; Orgel, L.E. Origins Life Evol. Biospheres 2002, 32, 99-102.
Yonemitsu, E.; Isshiki, T.; Kijima, Y. 1975, US Patent 4,059,582.
Costanzo, G.; Saladino, R.; Crestini, C.; Ciciriello, F.; DiMauro, E. BMC Evol. Biol. 2007,
7(Suppl 2), S1.