Student Manual
Student Protocol
Lab 1: SYNTHESIS OF ADENINE FROM CHEMICAL INVENTORY
Purpose: To synthesis adenine using chemical inventory.
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
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 your sample along with the standards of adenine, Thymine and cytosine
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 nitrogenous base that has been
synthesized.
Student Data Sheet for 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
4
8
9
11
Description of physical appearance
Sketch the TLC plates, before and after development.
Substance
Spot 1
Spot 2
Spot 3
Spot 4
Average
Calculations
Rf Value
IDENTITY OF UNKNOWN NITROGENOUS BASE: _____________________
Post-Lab Questions:
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.)
List the four nitrogenous bases that are found in DNA. Make sure to indicate which
bases are purines and which are pyrimidines.
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.
Which nitrogenous base did you hypothesize would be generated? Did your hypothesis
match your result?
Additional Questions (for Chemistry classes):
Identity and draw at least one functional group found in a molecule from the background.
Identify the number of sigma and pi bonds in the formamide structure (refer to
background section).
How do you think HCN was formed?
How many lone pairs of electrons are on each type of nitrogen atom in the DAMN
molecule?
Lesson:2- 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
Quantity
3
3
1
1
7
1
1 each
1
1
1
1
Quantity
1
1
Purpose:
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.
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 into the water. 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 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.
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 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
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:
1. On a scale from 1 – 5, how confident are you that your unknown’s were identified
correctly (5 being most confident)
2. What applications might this process have in other areas of science?
3. What were potential sources for error in this experiment?
.
Lab-3: STRAWBERRY DNA EXTRACTION LAB
Purpose: To extract the DNA from Strawberries.
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
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.
Background Information
In today’s lab, you will break apart the cells of a strawberry and release its DNA
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.
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
1
2
3
Purpose
Related Procedure Step(s)
4
5. Construct a concept map using at least 10 of the bold words found in the background.
Lab-4: DNA HYDROLYSIS LAB
Purpose: The purpose of this lab is to break apart a macromolecule, such as DNA, into
its monomers and smaller building blocks using water which is called Hydrolysis. Then
by using HCl acid cleave one of the bases from the sugar-phosphate backbone of DNA.
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:
Access to a 95C - 100C hot water bath
Access to a hair dryer
Access to a UV lamp
Quantity
1
1
1
1
1
4
1
1
1
1
1
4 ml
2ml
Quantity
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.
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.