Adenine Synthesis in a Model Prebiotic Reaction:
Adenine Synthesis in a Model Prebiotic Reaction:
Connecting Origin of Life Chemistry with Biology
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Lakshmi N. Anumukonda, Avery Young, David G. Lynn, Ragan Buckley, Amena Warrayat, Christina L. Graves, Heather D. Bean, and Nicholas V. Hud
NSF/NASA Center for Chemical Evolution
Adenine Synthesis in a Model Prebiotic Reaction:
Connecting Origin of Life Chemistry with Biology
Module Contents
1. Overview and Learning Objectives
a. Learning Objectives
i. National Standards
ii. Georgia State Standards
iii. International Baccalaureate Diploma Program Chemistry Standards
b. Laboratory Experiment Overviews
i. Experiment 1: Adenine Synthesis in a Model Prebiotic Reaction
ii. Experiments 2 and 3: DNA Extraction and Hydrolysis
iii. Experiment 4 (Optional): Thin Layer Chromatography
2. List of Chemicals and Supplies
3. Instructor’s Advance Preparation Guide and Protocol
a. Background Information
b. Laboratory Experiment Preparation Guides and Protocols
i. Experiment 1: Adenine Synthesis in a Model Prebiotic Reaction
ii. Experiment 2: Extraction of DNA from Strawberries
iii. Experiment 3: Hydrolysis of DNA
iv. Experiment 4: Thin Layer Chromatography
4. Student Manual
a. Background Information
b. Student Laboratory Experiment Guides
i. Experiment 1: Adenine Synthesis in a Model Prebiotic Reaction
ii. Experiment 2: Extraction of DNA from Strawberries
iii. Experiment 3: Hydrolysis of DNA
iv. Experiment 4: Thin Layer Chromatography
5. Appendices
a. Appendix A: Student Worksheets
i. Adenine Synthesis Worksheet
ii. DNA Extraction Worksheet
iii. Hydrolysis of DNA Worksheet
iv. Thin Layer Chromatography Worksheet
b. Appendix B: Teacher Answer Guides
i. Adenine Synthesis Answer Guide
ii. DNA Extraction Answer Guide
iii. Hydrolysis of DNA Answer Guide
iv. Thin Layer Chromatography Answer Guide
c. Appendix C: Chemical Safety Information
d. Appendix D: Glossary
e. Appendix E: References
Adenine Synthesis in a Model Prebiotic Reaction:
Connecting Origin of Life Chemistry with Biology
Overview and Learning Objectives
While many high school laboratory units are designed to help students understand the processes of natural selection and sexual selection, and the significance of mutation to evolution, few exist that allow students to investigate the chemistry of life’s origins. This series of inquiry-based laboratory experiments is designed to allow students to develop and utilize a useful set of laboratory skills while providing teachers with a foundation to incorporate prebiotic chemistry throughout both the biology and chemistry curricula.
Learning Objectives
National Standards: As a result of activities in grades 9-12,
• All students should develop
o Abilities necessary to perform scientific inquiry
o Understanding about scientific inquiry
• All students should develop an understanding of
o The structure of atoms
o The structure and properties of matter
o Chemical reactions
• All students should develop an understanding of
o The cell
o Biological evolution
o Matter, energy, and organization in living systems
Georgia State Standards:
This material may be taught not only during the units of chemistry and biology discussed below, but also a month or two before state graduation tests to refresh biology and chemistry concepts, enabling students to make broader connections between biology and chemistry and to improve graduation test scores.
International Baccalaureate Chemistry Standards:
Overview: These labs are also consistent with the IB Chemistry Curriculum, especially for teaching Organic Chemistry and the Option: Modern Analytical Chemistry (deducing the molecular and structural formulas of organic compounds; identifying sigma and pi bonds; identifying functional groups; calculating molar mass; determining the percentage composition of different elements; and understanding the purpose of, the physical phenomena underlying, and the applications of chromatography).
Experiment 1: Adenine Synthesis in a Model Prebiotic Reaction
Overview: In the first experiment of the series, students will conduct an experiment in which they synthesize adenine, one of the nitrogenous bases found in DNA, using chemicals that many scientists believe may have been present on the primitive Earth. This experiment will demonstrate a plausible prebiotic mechanism for the chemical formation of adenine, one of the components of DNA – one of life’s essential macromolecules.
Biology Objectives: Students will understand that chemical processes and materials available on the early Earth can generate the molecules that comprise living organisms. Students will also determine that the adenine in Experiment 1 is identical in nature to the biotic adenine extracted in Experiments 2 and 3 in this series.
Biology Curriculum Link: Evolution Unit
Biology GPS Standards: SCSh2, SCSh3, SCSh4, SCSh5, SB5
Chemistry Objectives: Students will perform a synthesis reaction in which they will generate adenine, using only chemicals that many scientists believe may have been present on the primitive Earth. Students will understand that chemical processes are responsible for creating biotic material. This experiment can also be used in higher-level chemistry classes to provide an example or extension for investigating organic chemistry.
Chemistry Curriculum Link: Types of Chemical Reactions Unit/Organic Chemistry Unit (Advanced Classes)
Chemistry GPS Standards: SCSh2, SCSh3, SCSh4, SCSh5, SC1
Duration: This experiment takes one 45-60 minute class period, depending on the availability of an oven or sand bath capable of reaching 180°C or 240°C, respectively. If the optional Experiment 4 demonstrating TLC techniques is not completed first, this lab may take longer as students will need to develop proficiency with the TLC technique.
Experiments 2 and 3: DNA Extraction and Hydrolysis
Overview: This three-part experiment takes a routine DNA extraction a step further, by allowing students to perform a DNA hydrolysis reaction in which one of the DNA nitrogenous bases (or nucleobases) is released. Students will use the thin layer chromatography (TLC) technique demonstrated by the teacher or developed in the optional fourth laboratory in this series to determine the identity of the unknown nitrogenous base.
Biology Objectives: Students will understand that DNA is a macromolecule found in all living things, and that nitrogenous bases are fundamental components of DNA. In addition, students will understand that DNA is a chemical structure that can be broken apart through hydrolysis reactions to release its individual components.
Biology Curriculum Link: DNA Structure/Function Unit
Biology GPS Standards: SCSh2, SCSh3, SCSh4, SCSh5, SB1, SB2
Chemistry Objectives: Students will understand that DNA is a chemical structure and will perform DNA hydrolysis, a type of decomposition reaction.
Chemistry Curriculum Link: Types of Chemical Reactions Unit
Chemistry GPS Standards: SCSh2, SCSh3, SCSh4, SCSh5, SC1, SC2
Duration: This experiment consists of three parts, each of which will take an entire class period. It is not essential that Part 1 be performed consecutively with Parts 2 and 3; it can be separated by up to two weeks. However, Parts 2 and 3 must be performed on consecutive days.
Experiment 4 (Optional): Introduction to Thin Layer Chromatography (TLC)
Overview: This experiment challenges students to identify unknown nitrogenous bases (or nucleobases). Students are introduced to TLC, which is used in the other laboratory experiments in this series. Alternatively, this experiment can be omitted and the teacher can provide a demonstration of the TLC technique for the class.
Biology Objectives: Students will become familiar with the nitrogenous bases that are found in DNA and will become proficient in spotting, developing, and analyzing TLC plates.
Biology Curriculum Link: Chemistry/Macromolecule Unit
Biology GPS Standards: SCSh2, SCSh3, SCSh4, SCSh5, SB1, SB2
Chemistry Objectives: Students will investigate one of the methods used for the separation of mixtures and will become proficient in spotting, developing, and analyzing TLC plates.
Chemistry Curriculum Link: Properties of Matter Unit
Chemistry GPS Standards: SCSh2, SCSh3, SCSh4, SCSh5, SC1
Duration: A 45-60 minute class period is sufficient to run the experiment. Additional class time may be spent teaching students how to calculate retention factors (Rf).
|Adenine Synthesis in a Model Prebiotic Reaction |
|Table S1: List of Chemicals and Supplies |
| | | | | |
|Fisher Scientific | | |
|VWR/Sargent Welch | | |
|Sorbtech | | |
|Flinn Scientific | | |
| | | | | |
|Reagent |Size |Vendor |Catalog Number |Pricea |
|Adenine | | | | |
| |25 g |VWR |AAA14906-14 |$47.60 |
| |50 g |VWR |AAA14906-18 |$88.10 |
| |10 g |Fisher |502303287 |$64.00 |
| |25 g |Fisher |AC14744-0250 |$73.10 |
| | | | | |
|Thymine | | | | |
| |25 g |VWR |AAAL04459-14 |$30.54 |
| |5 g |Fisher |AC15785-0050 |$30.40 |
| |25 g |Fisher |AC15785-0250 |$37.30 |
| | | | | |
|Cytosine | | | | |
| |5 g |VWR |AAA14731-06 |$36.10 |
| |25 g |VWR |AAAL09210-14 |$131.54 |
| |5 g |Fisher |AC16176-0050 |$47.80 |
| |25 g |Fisher |AC16176-0250 |$175.60 |
| | | | | |
|DAMN (Diaminomaleonitrile) | | | | |
| |25 g |VWR |AAB24328-14 |$20.50 |
| |100 g |VWR |AAB24328-22 |$53.10 |
| |25 g |Fisher |AC17270-0250 |$23.10 |
| |100 g |Fisher |AC17270-1000 |$66.70 |
| | | | | |
|Formamide | | | | |
| |500 g |VWR |EM-FX0421-4 |$103.07 |
| |1 L |VWR |EM-FX0421-6 |$135.89 |
| |1 L |Fisher |F84-1 |$57.11* |
| |500 g |Fisher |AC42374-5000 |$75.74* |
| | | | | |
|Ammonium Formate | | | | |
| |250 g |VWR |AAAL13166-30 |$19.73 |
| |1 kg |VWR |AAA10699-0B |$57.00 |
| |500 g |Fisher |A666-500 |$32.30* |
| |1 kg |Fisher |AC16861-0010 |$58.69* |
| | | | | |
|Item |Size |Vendor |Catalog Number |Pricea |
|1 Dram Vial (3.7 mL) | | | | |
| |144 vials |VWR |66011-041 |$82.26 |
| |144 vials |Fisher |03-339-21B |$55.26 |
| | | | | |
|8 Dram Vial (29.6 mL) | | | | |
| |144 vials |VWR |66011-165 |$69.22 |
| |144 vials |Fisher |03-339-21H |$90.90 |
| | | | | |
|Capillary Tubes | | | | |
| |100 tubes |VWR |CG184002 |$7.59 |
| |100 tubes |Fisher |80061-550 |$20.85 |
| | | | | |
|Thermometerb | | | | |
| |1 thermometer |VWR |89095-604 |$32.73 |
| |1 thermometer |Fisher |15-041-4F |$50.21 |
| | | | | |
|TLC Platesc | | | | |
| |50 plates (4 cm x 8 cm) |Sorbtech |1624187 |$41.76 |
| |25 plates (20 cm x 20 cm) |Sorbtech |1624126 |$150.00 |
| | | | | |
|UV Lamp (254 nm) 4 Wattd |1 lamp |VWR |82027-148 |$55.99 |
|UVP Tube (254 nm) 25 Watte |1 bulb |Fisher |UVP 34007301 |$48.48 |
|HCl (3 M or stronger) | |Flinn | | |
|Sodium Chloride | |Flinn | | |
|Filter Paper | |Flinn | | |
|Beakers | |Flinn | | |
|Watch Glasses | |Flinn | | |
|Balance | |Flinn | | |
|Ruler | |Various | | |
|Scissors | |Various | | |
|Rubber Bands | |Various | | |
|Plastic Wrap | |Various | | |
|Pencilsf | |Various | | |
|Strawberries | |Various | | |
|Clear Soap | |Various | | |
|Toothpicksg | |Various | | |
|Ethanol | |Various | | |
|Test Tubes | |Various | | |
|Test Tube Tongs | |Various | | |
|Test Tube Racks | |Various | | |
|10 mL Graduated Cylinder or Transfer Pipettes | |Various | | |
|Hair Dryer | |Various | | |
|Test Tube Stopper, Aluminum Foil, or Parafilm | |Various | | |
|Cheesecloth, Coffee Filters, or Kimwipes | |Various | | |
|Glass Stirring Rods | |Various | | |
|Tweezers or Forceps | |Various | | |
|Hot Plate | |Various | | |
|Distilled Water | |Various | | |
Notes:
a) Prices are subject to change and discounts may be available depending on your county, state, or school district. Prices marked with an asterisk (*) are Georgia state contract prices and may be different in other locations. Part numbers are correct as of date of this publication but are also subject to change; when in doubt in the case of reagents, refer to the CAS numbers given in the safety section to ensure you are ordering the correct product.
b) Make sure not to exceed the temperature limit of the thermometer you use. The ones listed are non-mercury thermometers that read up to 260°C.
c) TLC plates are available from Sorbtech and other manufacturers in a variety of package sizes, plate sizes, and price ranges. If you choose to purchase plates on your own, you should choose plates with silica adsorbent and a 254 nm UV indicator. Polyester-backed plates can be cut with scissors to smaller sizes and are lower in cost than glass-backed plates. Some vendors will offer quotes with reduced prices if contacted before the purchase.
d) This lamp requires 4 AA batteries (not included).
e) This is a UV-emitting fluorescent tube only. It will fit into the hood portion of a 10-gallon aquarium.
f) Pencils must be non-mechanical. (Mechanical pencils damage the surface of the TLC plates.)
g) Toothpicks are listed as an alternative to capillary tubes. If capillary tubes are purchased, toothpicks are not needed.
Adenine Synthesis in a Model Prebiotic Reaction:
Connecting Origin of Life Chemistry with Biology
Instructor’s Advance Preparation Guide and Protocol
Background Information
Billions of years ago, Earth existed only as a cloud of gas, which was so hot that even the simplest compounds could not exist. Under the influence of gravity, atoms that made up the primordial gas cloud would have begun to organize into layers, with the densest gases at the center and the least dense ones on the outer edges. As the cloud cooled, its innermost components condensed into a liquid, while the outer layers remained in the form of an envelope of gas.
Further cooling allowed for the formation of molecules: simple ones formed first, and then reacted with one another to form more complex molecules. Gradually, the Earth’s crust developed from a solid envelope of condensed compounds surrounding the hot core of the planet.
As the molten center of the Earth continued to cool, it contracted. The rigid layer above, unable to contract alongside the liquid center, formed cracks and folds through which molten rock poured out onto the surface of the planet. Modern geologic events, such as volcanic eruptions and mountain building, are evidence that these same phenomena still occur today.
At first, the Earth’s primitive atmosphere consisted of just those atoms found in the outer portion of the initial gas cloud. A combination of atmospheric cooling and volcanic activity generated an even more diverse set of molecules. While the exact composition of the Earth’s primitive atmosphere is not known, various models suggest that it contained water (H2O), carbon dioxide (CO2), methane (CH4), ammonia (NH3), and molecular nitrogen (N2). Formamide (H2NCOH) and hydrogen cyanide (HCN) were also likely present among the early molecules on Earth, and are believed to have played an important role in the chemical processes that produced the first building blocks of life, including amino acids and nucleobases.
Finally, when the Earth’s temperature fell to 100°C, it became possible for water in the atmosphere to exist in the form of liquid droplets. Continuous downpours of rain fell upon the surface of the planet, inundating it and forming an ocean. Organic molecules formed in the atmosphere dissolved into the liquid drops and accompanied them to the Earth’s surface. These compounds then encountered each other and reacted to form larger molecules, including the polymers required for the emergence of life (e.g. peptides and oligonucleotides). For additional background information on origin of life chemistry and early chemical evolution, instructors are referred to the website of the Center for Chemical Evolution () and to the book by Wills and Bada, The Spark Of Life: Darwin And The Primeval Soup (1).
Experiment 1: Adenine Synthesis in a Model Prebiotic Reaction
In this experiment, you will be using materials that were likely available on the early Earth to generate and identify a nitrogenous base of DNA and RNA using thin layer chromatography (TLC). The reactants used in this lab are formamide, ammonium formate, and diaminomaleonitrile (DAMN). The likely availability of formamide on the early Earth is supported by the discovery of this molecule in comets and other interstellar objects (2). The other reactants – ammonium formate and DAMN – can readily be generated from either formamide or hydrogen cyanide (HCN).
The reaction below illustrates how HCN can be hydrolyzed to form formamide, which can further be hydrolyzed to ammonium formate:
[pic]
Scheme S1: Chemical equilibria between HCN, formamide, and ammonium formate.
DAMN is formed from four HCN molecules in concentrated solutions of HCN (3). There are several chemical intermediates in this process, although the precise mechanism is currently unknown:
[pic]
Scheme S2: 4 molecules of HCN polymerize to form diaminomaleonitrile, or DAMN.
Preparation Overview:
• TLC plates should be cut with the dimensions 2" × 3" – 15 minutes (exact measurement is not important, but care must be taken that the edges are straight with right angles at the corners)
• Preheat your oven to 180°C or your sand bath to 240°C – 20 minutes
• Prepare standard solutions of adenine, thymine, and cytosine: Each group will use a very small amount of each of the nitrogenous base stock solutions, so a few milliliters of each can accommodate many students. For long periods of storage, freezing is preferred to limit degradation. To prepare stock solutions of adenine, thymine, and cytosine, add 0.1 g of base to approximately 100 mL of water. If this is not sufficient to dissolve the entire added base, add water in increments of 20 mL at a time until the white solute has completely dissolved. Note: guanine is not used as a nitrogenous base in this laboratory experiment series because it has very low solubility in water and requires a different mobile phase in the chromatography chamber.
• Prepare TLC developing chambers (using a beaker or plastic cup, watch glass or plastic wrap, filter paper, and rubber band if using plastic wrap) – the chamber just needs to be large enough to accommodate the width and height of the TLC plate – 15 minutes
• Prepare capillary tubes: One long capillary tube can be separated into two smaller tubes with narrow openings by using the heat from a Bunsen burner or a candle. To accomplish this, hold the opposite ends of the capillary tube in your hands and place the center over a flame. When the glass starts to soften, pull the capillary tube apart in one quick motion. Gently tap the melted end onto a hard surface to remove any oddly-shaped, bent, or closed points. Note: Toothpicks can be used instead of capillary tubes; however, students often make too large of a spot with toothpicks. Flat toothpicks are preferable to thicker, rounded ones.
• Obtain hair dryers: 2-3 hair dryers should be sufficient for the laboratory. 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.
The actual amounts of ammonium formate, formamide, and DAMN to be used can be changed depending on the stock available. However, a 1 g: 6 mL: 0.1 g ammonium formate: formamide: DAMN ratio is required. Accurate measurements by the students are very important; however, the amount of reaction mixture produced by the quantities listed in the lab protocol is much greater than required for student use, even when the products of one reaction are shared among the entire class. If equipment capable of measuring smaller quantities is available, it is recommended that reduced quantities of reactants be used. Another method for reducing the quantities of chemicals required is to perform the reaction as a demonstration, and then to divide the reaction mixture into portions for each group of students to spot on their TLC plates.
Teacher’s note: If desired, a control reaction may also be included in this experiment. For example, a quantity of the DAMN reaction mixture could be removed before heating, and spotted in an additional lane to show that heating is required for the production of adenine. Alternatively, the formamide and ammonium formate mixture could be heated in the absence of DAMN, to show that DAMN is a crucial component in the synthesis of adenine. If either of these options is chosen, the student instructions would need to be changed accordingly.
|Table S2: Required Materials for 8 Lab Stations |Quantity |
|Formamide |48 mL |
|Ammonium formate |8.0 g |
|Diaminomaleonitrile (DAMN) |0.8 g |
|Capillary tube/toothpick |32 |
|Vials containing prepared solutions of adenine, cytosine, and thymine |8 each |
|Test tube |8 |
|50 mL beaker |8 |
|Thermometer |8 |
|Spatula |8 |
|Weigh boat |16 |
|Test tube tongs |8 |
|Test tube rack |8 |
|Glass stirring rods |8 |
|Distilled water | |
|10 mL graduated cylinder or a transfer pipette |8 |
|TLC plate |8 |
|TLC developing chamber |8 |
|Ruler |8 |
|Pencil (non-mechanical) |8 |
|Tweezers or forceps |8 |
|Table S3: Common Workstation |Quantity |
|Short wave (254 nm) UV lamp |1 |
|Hair dryer |1 |
|Access to an oven or sand bath |1 oven or 2-3 sand baths |
|Balance |1 |
Teacher’s note: a sand bath can be made from any type of sand. You can purchase the sand from a craft or home improvement store. The number of students using the sand bath will determine the size of the sand bath required. A 500 mL beaker sand bath can accommodate at least five test tubes. The actual volume of sand used is not important as long as the tubes are covered up to the level of the reagents. A sand bath large enough to accommodate an entire class can also be created using a large hot plate and a Pyrex dish or deep aluminum pan.
|Table S4: Student Workstation |Quantity |
|Formamide |6 mL |
|Ammonium formate |1 g |
|Diaminomaleonitrile (DAMN) |0.1 g |
|Prepared solutions of adenine, cytosine, and thymine |1 of each |
|Capillary tube/toothpick |4 |
|Test tube |1 |
|50 mL beaker |1 |
|Thermometer |1 |
|Spatula |1 |
|Weigh boat |2 |
|Test tube tongs |1 |
|Test tube rack |1 |
|Glass stirring rod |1 |
|10 mL graduated cylinder or transfer pipette |1 |
|TLC plate |1 |
|TLC developing chamber |1 |
|Ruler |1 |
|Pencil |1 |
|Tweezers or forceps |1 |
Procedure
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Step 1: Obtain and label a clean test tube with your initials.
Note: Ammonium formate, formamide, and DAMN are irritants to the skin and eyes. Wear gloves and use caution when handling these chemicals. If one of these chemicals comes in contact with your skin, wash the affected area immediately with soap and water.
Step 2: Using one of your weigh boats, measure 1.0 g 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 stopper your test tube, and place it into the sand bath (or oven) for 5 minutes. Record the time on your data sheet and calculate when you will need to remove your sample; record that time on your data sheet, as well. Proceed to Step 6 while your sample is in the sand bath.
Step 6: Using your other weigh boat, measure exactly 0.1 g of the diaminomaleonitrile (DAMN) and set aside for use in Step 8.
Step 7: After 5 minutes, 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 it to the sand bath for an additional 3 minutes before moving to Step 8.
Step 8: Record the physical appearance of your solution on your data sheet. Once your test tube is cool to the touch, carefully add the DAMN you measured in Step 6 into your test tube and thoroughly mix with your 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 so that all DAMN comes off 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 or oven for 20 minutes. Record the time of entry on your data sheet and calculate when you will need to remove your sample; record that time on your data sheet as well. While your test tube is in the sand bath, prepare your TLC plate and chamber. You will need room for four spots on your plate – your reaction sample and the stock solutions of adenine, cytosine, or thymine.
To prepare your TLC developing chamber:
1. Obtain a developing chamber from the front of the room. The developing chamber consists of a beaker or plastic cup, a watch glass or piece of plastic wrap, a piece of filter paper, and, if using plastic wrap, a rubber band.
2. Place the filter paper into the chamber, as shown in the image below. You may need to cut the paper to a smaller size to fit into the chamber. The paper should be taller than the TLC plate and the width of the paper should cover no more than half the surface inside the chamber. The filter paper ensures that the inside of the chamber will remain saturated with vapor from the aqueous mobile phase, so that the TLC plate will run correctly.
[pic] [pic]
Figure S1: Examples of TLC chambers.
3. Carefully pour water into the developing chamber to a depth of approximately 0.5 cm. Water will begin to travel up the filter paper. Do not use the TLC chamber until the water completely saturates the filter paper. If you wish to speed up this process, you can carefully tip the chamber to wet the filter paper.
To prepare your TLC plate:
1. Using a ruler and a pencil (do not use a mechanical pencil or a pen), draw a line across the TLC plate 2.0 cm from the bottom, as indicated in the picture below. Press the pencil lightly so as not to damage the coating on the TLC plate. This line will serve as the origin line.
2. Pressing gently, draw four hash marks across the origin line (see an example hash mark in the picture below). Space these evenly across the origin line, but start and end at least 0.5 cm from the left and right sides of the plate. Below the origin line, pressing lightly with your pencil, label hash marks with A, C, T, and R (for adenine, cytosine, thymine, and your reaction mixture).
[pic]
Figure S2: TLC plate with hash marks and origin line marked.
Step 11: After 20 minutes, use your test tube tongs to remove your sample from the sand bath or oven 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 into a small beaker. Place the remaining 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 beaker containing reaction mixture, and stir.
Step 14: Spot your TLC plate according to the directions below.
To spot a TLC plate:
1. You will be using capillary tubes (or toothpicks) to spot your adenine, cytosine, and thymine standards on the TLC plate, as well as your reaction mixture. Each solution will require a separate capillary tube to prevent cross-contamination. Label the top of each capillary tube with a piece of tape, using the same letters you used to label the hash marks on the TLC plate. Use caution with the capillary tubes, as they are fragile and very sharp when broken.
2. Take the capillary tube labeled A and insert the sharpened end into your stock solution of adenine. You should be able to see the solution rise up into the tube, through capillary action. If you are using toothpicks, dip a toothpick into the adenine solution and slowly remove it; you want a very small drop on the end.
3. Touch the end of the capillary tube gently on the origin line at the hash mark 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 tube run onto the TLC plate. You will not use all of the solution inside the tube. If you are using toothpicks, you will spot the entire drop of adenine solution on the plate. Touch your toothpick gently to the hash mark on the origin line, being careful not to damage the surface of the TLC plate. Make sure the spot is small.
4. Repeat this process for the cytosine and thymine solutions.
5. Insert the sharpened end of the final capillary tube into the beaker where you diluted your reaction product mixture with water. Spot this sample in the final location (labeled R) on your origin line. (If using toothpicks, follow the instructions in #2 and #3 above for applying samples to the toothpick and spotting on a TLC plate.)
6. Allow the spots on the TLC plate to dry completely. (You will not be able to see the spots on the origin line when they are dry.) You may blow gently on the TLC plate to speed up the drying process. If you would like to check your work, to see if you have spotted enough of your solutions onto the TLC plate, you can look at it under the shortwave UV lamp. You should see dark spots on the origin line where you placed your compounds. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step 15: Develop your TLC plate according to the directions below.
To develop a TLC plate:
1. Before placing your TLC plate into the developing chamber, measure and compare the height of the water in relation to the origin line on your TLC plate. If it appears that the water level of the chamber will be above your origin line when you place the 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 obtain and spot a new TLC plate. If you are unsure whether the water will cover the line, err on the side of caution and remove some of the water. You can always add water back with no negative effects if there is not enough water in the beaker to develop the plate.
2. Without touching the plate directly with your fingers, use your tweezers to carefully place the prepared TLC plate in the developing chamber so that its bottom edge is sitting flat on the bottom of the chamber and leaning against the side of the chamber that is not covered by the filter paper. Be very careful! If the plate falls into the water, you will have to obtain and spot a new TLC plate. Make sure you record the time when you first placed the plate into the chamber.
3. Cover the chamber with the plastic wrap or watch glass. If using plastic wrap, secure the plastic wrap over the chamber opening with a rubber band, making sure not to move the TLC plate within the chamber. If your plate falls over, you can use your tweezers to put it back into position, provided water did not get on or above the origin line. DO NOT pick up the chamber once your plate is in place and running.
4. Watch as the mobile phase runs up the TLC plate. When the mobile phase is approximately 1 cm from the top of the plate, remove it from the chamber using your tweezers and place it face up on a paper towel. DO NOT allow the mobile phase to overrun the top of your plate.
5. Using a pencil, trace the location of the mobile phase on your plate. This is called the solvent front, and marking the solvent front is a critical step for calculation of the retention factor (Rf). Note, the solvent front is sometimes not a straight line, and may be higher in the center of the plate than at the edges. This is why we make our hash marks at least 0.5 cm away from the edges of the plate.
[pic]
Figure S3: TLC plate that has been developed, indicating the position of the solvent front.
6. Once you have traced the solvent front, hold your TLC plate with your tweezers and use the blow dryer to completely dry the plate.
Step 16: Visualize the spots on your TLC plate. To do this, place your TLC plate underneath a downward facing shortwave UV lamp. Mark all spots that you see, no matter how faint, with a pencil, tracing their outlines. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step 17: Calculate the Rf of each spot according to the directions below. Use this information to identify the unknown nitrogenous base that has been synthesized by matching the Rf of your main product (largest spot) in your reaction to the Rf of one of the three standards. A given compound will always have the same Rf under the same conditions (e.g. stationary phase, mobile phase); this property is thus frequently used to identify unknown compounds.
Sample Rf calculation: The calculation below is for a single compound (spot) in a sample that is a mixture of substances, and therefore produces more than one spot on the TLC plate. The procedure for determining the Rf for each spot within one sample is exactly the same as the procedure for determining the Rf value for many individual samples on one plate.
[pic]
Figure S4: Illustration of a TLC plate including sample spots, origin line, and solvent front, to be used as an aid for calculating Rf.
The solvent front is the distance the mobile phase traveled on the plate, and is what you recorded by a pencil line when you took your TLC plate out of the developing chamber. Make sure you measure from the center of each spot to the origin line to obtain the distance moved by the molecule.
The formula for calculating Rf is:
Rf = distance moved by the molecule / distance moved by the solvent front
The Rf for the substance indicated by the asterisk (*) would be:
Rf = 5.5 cm/6.0 cm = 0.92
Experiment 2: Extraction of DNA from Strawberries
Background Information
In today’s experiment, you will break apart the cells of a strawberry and isolate the DNA (deoxyribonucleic acid) in a form that is visible to the naked eye. Wild strawberries are diploid, meaning they contain two copies of each chromosome. Commercially produced strawberries are octoploid. Because they contain eight copies of each chromosome, they are particularly good to use for this lab.
The DNA in today’s experiment will precipitate, or come out of solution, as long white strands. The white material is actually thousands, or even millions, of DNA strands (and associated proteins) wrapped around each other. An individual strand of DNA is so small that it can only be imaged using the most specialized microscopes. The “images” that helped Watson and Crick to uncover the double helix structure of DNA were actually diffraction patterns acquired by Rosalind Franklin, who passed X-rays through DNA fibers, much like the fibers of DNA that you will isolate today.
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 step is accomplished, in part, by the physical act of smashing the strawberries in a Zip-top bag. Ripened fruit is also used in this lab because the cell walls are already weakened during 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 the membranes while leaving the DNA intact.
The third step is the separation of the DNA from the coarse, 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 and therefore, not visible in the filtered strawberry mixture. However, DNA is insoluble in ethanol in the presence of salt. Thus, the careful addition of ethanol onto the top of the DNA mixture will cause the DNA to come out of solution and form visible, insoluble threads that can be spooled and collected.
Preparation Overview:
• Create extraction buffer. The DNA extraction buffer is made from water, soap, and sodium chloride (NaCl). It is preferable to use a clear soap that is free of aloe, fragrances, lotions, and other additives. For advanced classes, it is recommended that students make their own buffer solutions. 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 solution as necessary. Note: You do not want the extraction buffer to be very sudsy. The buffer will also keep for long periods of time. – 15 minutes
• Chill ethanol by putting it into an ice-water bath or a freezer for at least 30 minutes prior to the beginning of class. Note: 95-100% ethanol is preferable although concentrations down to 70% ethanol may be used. If a lower concentration of ethanol is used, increase the volume of ethanol or decrease the volume of water accordingly; you will want approximately three times as much ethanol as water in the precipitation step. – 30 to 45 minutes
|Table S5: Required Materials for 8 Lab Stations |Quantity |
|Strawberries |8 to 10 |
|Zip-top bags |8 |
|Water |500 mL |
|Clear lab soap |5 mL |
|Sodium chloride (NaCl) |5 g |
|250 or 100 mL beakers |8 |
|Additional 100 mL beakers |8 |
|Cheesecloth/coffee filters/Kimwipes |Box |
|Test tubes |16 |
|Test tube stoppers/Parafilm/aluminum foil |8 |
|10 mL graduated cylinders |8 |
|Ice cold ethanol |400 mL |
|Stirring rods |8 |
|Transfer pipettes |8 or 16 |
|Table S6: Student Workstation |Quantity |
|Strawberry |1 |
|Zip-top bag |1 |
|DNA extraction buffer |25 mL |
|250 or 100 mL beaker |1 |
|Additional 100 mL beaker |1 |
|Cheesecloth/coffee filter/Kimwipe |5 |
|Test tubes |2 |
|Test tube stopper/Parafilm/aluminum foil |1 |
|10 mL graduated cylinder |1 |
|Ice-cold ethanol |50 mL |
|Stirring rod |1 mL |
|Transfer pipette |1 or 2 |
Procedure
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Step 1: Remove the green stem from one strawberry and place the strawberry into a plastic zip-top bag.
Step 2: Remove the air from the bag, seal it, and mash the strawberry for two minutes, 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 one minute or until the solution is thoroughly mixed with the 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 the 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 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 the 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 the DNA precipitate out of solution as an opaque white substance at the interface between the ethanol and the filtered solution. Occasionally, the DNA may precipitate as a clear substance, which can be seen because of bubbles formed, but it will still be at the interface of the ethanol and the filtered solution. Make a sketch of what you see and record your observations of the precipitated DNA on your data sheet.
Step 11: Allow the DNA solution to sit undisturbed for five minutes. Remove the DNA that has accumulated at the surface by spooling it with a stirring rod or transfer pipette and place it into a test tube. If using a transfer pipette, move as little of the ethanol as possible when transferring the DNA.
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 will be DNA.
Step 13: Fill a 1 mL transfer pipette with water, and add about 0.5 mL 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 to your sample without your teacher’s approval. Record the amount of water used on your data sheet.
Note: Dissolving the DNA should cause the solution to become thicker and cloudier, and most of the white strands of the DNA will disappear.
Step 15: Place a stopper into the test tube, label your test tube, and place it into the designated location.
Experiment 3: Hydrolysis of DNA
Background Information
The DNA within all living organisms, from bacteria to humans, is made up of the exact same chemical components: deoxyribose, phosphate, and four nitrogenous bases: adenine (A), thymine (T), cytosine (C), and guanine (G). The difference in the amount of DNA and the specific arrangement of the nitrogenous bases (i.e. the genetic code) is what accounts for the wide variation of life forms on Earth. In very similar organisms, such as in humans and other primates, the information encoded by DNA is almost identical. Comparing the similarities between the DNA sequences (i.e. genomes) of two species, or two individuals of the same species, can help scientists determine the relatedness of two species or individuals.
The primary structure of DNA is generally described in terms of nucleotides, the molecular unit defined as a nucleobase connected by a glycosidic bond at the 1′ position of a phosphorylated 2′-deoxyribose sugar (Figure S5). Along the backbone of a DNA polymer, or strand, nucleosides are connected to each other through the phosphate groups. A phosphodiester linkage exists between the 3′ position of each nucleoside and the 5′ position of the next nucleoside along a given DNA strand (Figure S5). The order of the nucleotide bases (A, G, C and T) along a strand is the chemical basis for coding the genetic information contained within a DNA polymer. The repeating 3′-phosphate-5′ linkages between nucleotides provide the polymer directionality for DNA.
In eukaryotic cells, DNA is contained within the nucleus and the mitochondria, as well as in the chloroplast of photosynthetic organisms. In bacteria, DNA is contained within a less well-defined structure known as the nucleoid that, unlike the eukaryotic nucleus, is not surrounded by a membrane. The secondary structure of DNA within living organisms is predominantly a double helix formed by two strands with complementary nucleoside sequences, that is, sequences which support Watson-Crick base pairing along their entire lengths (i.e. A paired with T; G paired with C). The two strands of a Watson-Crick double helix run in opposite directions (as defined by the directionality of their polymer backbones) (Figure S5). Notice how the sugar molecules in Figure S5 below are oriented differently on the opposite sides of the paired bases. A Watson-Crick duplex is therefore said to have an antiparallel arrangement of its two strands. This twisted structure of two base-paired DNA polymers is the reason that DNA is commonly referred to as a double helix.
[pic][pic]
Figure S5: DNA Structure. (Left) The chemical structure of DNA. The bond indicated by a line between the nitrogen atom of adenine (A) and the 1´ carbon of the deoxyribose sugar is the bond that is broken during the depurination reaction that releases adenine from the DNA backbone. (Right) A schematic representation of the DNA double helix emerging from a chromosome. Right image courtesy of Genome Management Information System, Oak Ridge National Laboratory. .
The four nucleobases of DNA, adenine (A), thymine (T), cytosine (C), and guanine (G), are of two chemical classes: the purines (A and G) and the pyrimidines (T and C) (Figure S6). As shown above in Figure S5, in a DNA duplex, each base pair is formed by the hydrogen bonding of a purine nucleobase with a pyrimidine nucleobase. That is, adenine forms a base pair with thymine and guanine forms a base pair with cytosine.
[pic]
Figure S6: The chemical structures of the nucleobases of DNA.
Hydrogen bonds are not only important for the formation of Watson-Crick base pairs, but are also the bonds that hold together liquid water and are the bonds used within living cells to guide a multitude of molecular interactions. Thus, it is important to appreciate the nature of hydrogen bonds. 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 a single water molecule. In the case of water, the oxygen is more electronegative, or has a higher attraction for the shared electrons than does the 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 and a partially negative oxygen or nitrogen atom in neighboring molecules. Hydrogen bonds involving both oxygen and nitrogen atoms are responsible for base pairing in DNA (Figure S5).
In today’s experiment, you will be using the DNA extracted in the previous laboratory, and you will treat it with heat and acid to release one of its nitrogenous bases. Since the nitrogenous bases all have slightly different chemical structures, the bond that attaches them to the deoxyribose of the backbone varies in strength. The process of breaking apart a macromolecule like DNA into its monomers and smaller building blocks is called hydrolysis, and requires the use of water. The breaking of a bond by hydrolysis can be seen as the opposite of the formation of the same bond, which results in the production of a water molecule. Bonds that can be hydrolyzed include those that hold together the amino acids in proteins (the peptide bonds) and the nucleotides of DNA (the nucleobase-deoxyribose glycosidic bonds, and the phosphate-deoxyribose phosphate-ester bonds). Such bonds are often called reversible bonds, which allow living organisms to recycle the building blocks of proteins and nucleic acids.
To prepare the isolated genomic DNA for partial hydrolysis (that is, release of a nucleobase), the DNA is first dissociated from proteins, called histones, which package the DNA into its chromosomal structure. Separating DNA from its bound histones is accomplished by heating. Secondly, a strong acid, such as the HCl used in this experiment, is required. 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 proton, to the purine bases, which catalyzes the cleavage of the bond that holds these nucleobases to the sugar-phosphate backbone. After partially hydrolyzing the DNA, you will use thin layer chromatography (TLC) to determine the identity of the base that has been liberated from the polymer by cleavage of its bond with deoxyribose (the glycosidic bond).
Preparation Overview:
• Prepare 3M hydrochloric acid from a more concentrated stock: 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 water. When diluting an acid, always add acid to water, not the reverse. – 10 minutes
• Prepare adenine, cytosine, and thymine standard solutions: If the “Adenine Synthesis” experiment was completed previously, and the solutions of these nitrogenous bases are only a week or two old, or have been frozen, the solutions from that experiment can be used. Otherwise, refer to the instructions on preparing these solutions from the “Adenine Synthesis” experiment.
• Prepare capillary tubes: Refer to the instructions from the “Adenine Synthesis” experiment regarding the pulling of capillary tubes.
• Prepare TLC chambers: Refer to the instructions from the “Adenine Synthesis” experiment.
• Obtain hair dryers: See note from “Adenine Synthesis” experiment.
• Pre-heat a water bath: To make sure there is sufficient time to heat the samples, it is recommended that the hot water bath be turned on and brought up to a 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 of the experiment.
Teacher’s note: This experiment can be completed in two 45-60 minute laboratory periods, which need to occur on successive days.
|Table S7: Required Materials for 8 Laboratory Stations |Quantity |
|DNA solution prepared in DNA Extraction lab | |
|Test tube |8 |
|Pair of test tube tongs |8 |
|Test tube rack |8 |
|Rubber stopper |8 |
|TLC plate |8 |
|TLC chamber |8 |
|Capillary tubes/toothpicks |32 |
|Ruler |8 |
|Pencil (not mechanical) |8 |
|Pair of forceps |8 |
|10 mL graduated cylinder (or 3 transfer pipettes) |8 |
|3M HCl |32 mL |
|Distilled water | |
|Thermometer |8 |
|500 mL beaker |8 |
|25 mL beaker |8 |
|Hot plate |8 |
|Table S8: Common Workstation |Quantity |
|Access to a 95°C – 100°C hot water bath |1 or 2 |
|Access to a hair dryer |1 or 2 |
|Access to a short wave (254 nm) UV lamp |1 |
|Table S9: Student Workstation |Quantity |
|DNA solution prepared in DNA extraction lab | |
|Test tube |1 |
|Pair of test tube tongs |1 |
|Test tube rack |1 |
|Rubber stopper |1 |
|TLC plate |1 |
|Capillary tubes/toothpicks |4 |
|Ruler |1 |
|Pencil (non-mechanical) |1 |
|Pair of tweezers or forceps |1 |
|TLC chamber |1 |
|10 mL graduated cylinder (or 3 transfer pipettes) |1 |
|3M HCl |4 mL |
|Distilled water | |
|Thermometer |1 |
|500 mL beaker |1 |
|25 mL beaker |1 |
|Hot plate |1 |
Procedure:
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Part 1
Step 1: Obtain a 500 mL beaker and a thermometer. Fill the beaker with 350 mL of water and place it onto the hot plate. Turn the hot plate 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 2 mL of your extracted DNA solution and place it into your labeled test tube.
Step 4: Cover the test tube. Check the temperature of your hot water bath. When it 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.
Note: Hot plates may vary. If your water bath begins boiling, turn it down slightly. If, after some time, it has not reached 95°C, you may need to turn the temperature up.
Step 5: Heat the sample for 30 minutes, 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 minutes for 30 minutes.
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.
Step 7: Allow your test tube to cool to the touch. Carefully add 2 mL of water and 4 mL of 3M HCl to your DNA solution. NOTE: HCl is a strong acid. Use caution when handling this substance, and wash your skin immediately with water for several minutes if it spills on you.
Step 8: Cover your test tube and place it back into the 95°C hot water bath. Record the time. Measure the temperature every 10 minutes and record that information on your data sheet.
Step 9: After 30 minutes, use your test tube tongs to remove your test tube from the water bath. Put it in the storage location designated by your teacher. If your class period will be over in less than 30 minutes, your teacher will complete this step for you.
Teacher’s note: For a 45-60 minute class, you may need to remove students’ test tubes from the water bath after the HCl treatment. For a block schedule, students will be able to do this step themselves.
Part 2
Step 1: Obtain your hydrolyzed DNA solution from the previous laboratory period. Record the appearance of your DNA solution after heating and treatment with HCl.
Step 2: Prepare your TLC plate and chamber. If you do not remember how to do this, please refer to the instructions from the “Adenine Synthesis” laboratory. You will be running four samples on your TLC plate – your hydrolyzed DNA and the adenine, cytosine, and thymine standard solutions you used in the “Synthesis of Adenine” lab.
Step 3: 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 4: Spot the adenine, cytosine, and thymine standard solutions onto your plate at the appropriate locations. Make sure your spots are completely dry, and then run your TLC plate in the chamber. Refer to the instructions from the “Adenine Synthesis” laboratory if you do not remember how to do this step.
Step 5: When the mobile phase is approximately 1 cm from the top of the plate, remove it from the chamber and immediately trace the location of the solvent front with a pencil.
Step 6: Dry your plate using a hair dryer, and use the UV lamp 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 the light facing down at all times.
Step 7: Calculate the Rf of your sample and of the adenine, cytosine, and thymine standards. Identify the nitrogenous base liberated from your DNA sample. If you do not remember how to calculate Rf, please refer to the “Adenine Synthesis” experiment.
Experiment 4 (Optional): Thin Layer Chromatography
Background Information
Chromatography is a method of separating and identifying chemicals in mixtures containing two or more compounds. The separation is accomplished by the distribution of the components of the mixture between two phases: one that is stationary and one that moves, or is mobile. A variety of solid stationary phases exist, and the mobile phase can be liquid or gas. Different compounds will have different solubilities and interactions with the stationary and mobile phases; chromatography takes advantage of these properties to allow for the separation of complex mixtures of compounds.
Thin layer chromatography, or TLC, is a technique in which the stationary phase is solid and the mobile phase is liquid. The stationary phase that you will use in today’s laboratory is silica gel that covers and is supported by a plastic backing. Alumina is another common stationary phase used in TLC. The mobile phase you will use is water; depending on the compounds you wish to separate by TLC, many other mobile phases are available.
TLC is commonly used to monitor whether organic reactions have proceeded to completion and to test purification conditions for synthesized organic compounds. It is also a useful technique for learning about a mixture of unknowns – for example, determining the number components present in a mixture and their identities.
The different distance each molecule travels along the adsorbent (stationary phase) in relation to how far the mobile phase has traveled is called the retention factor (Rf) and can be used to identify an unknown.
This experiment is designed to build your TLC skills. In the other experiments of this four-part series, you will use TLC to identify a nitrogenous base that you have synthesized in a model prebiotic reaction, and a nitrogenous base that you have hydrolyzed from strawberry DNA.
Preparation Overview
Teacher’s note: If class time is limited, you may choose to omit this experiment and instead do a demonstration of TLC techniques for the class before they complete the other laboratories in this series. If you feel that your class would benefit from TLC practice, then this laboratory can be done prior to the “Adenine Synthesis” laboratory.
• TLC plates should be cut with the dimensions 2" × 3" – 15 minutes (exact measurement is not important but care must be taken that the edges are straight with right angles at the corners)
• Prepare standard solutions of adenine, thymine, and cytosine: Each group will use a very small amount of each of the nitrogenous base stock solutions, so a few mL of each can accommodate many students. Freezing for long periods of storage is preferable, as the nitrogenous bases can degrade over long periods of time in aqueous solution. To prepare stock solutions of adenine, thymine, and cytosine, add 0.1 g of base to approximately 100 mL of water. If this amount of water is not sufficient to dissolve the entire added base, add water 20 mL at a time until the white solute has completely dissolved. Note: guanine is not used as a nitrogenous base in this laboratory series because it has low solubility in water and requires a different mobile phase in the chromatography chamber.
• Prepare “unknown” solutions: Four unknown solutions will be used in this experiment. For Unknown 1, use any one of the standard solutions of adenine, cytosine, and thymine. For Unknown 2, use any other standard solution. For Unknown 3, use a mixture of any two standard solutions (for example, adenine and thymine). For Unknown 4, use a different mixture of two standard solutions (for example, cytosine and thymine). For the mixtures, simply mix equal volumes of the two standard solutions being used. – 15 minutes
• Prepare TLC developing chambers (beaker or plastic cup, watch glass or plastic wrap, filter paper, rubber band if using plastic wrap) – the chamber just needs to be large enough to accommodate the width and height of the TLC plate. If not enough beakers are available to prepare 16 chambers, students can run their plates one after another in the same chamber. – 15 minutes
• Prepare capillary tubes: One long capillary tube can be separated into two smaller tubes with narrow openings by using the heat from a Bunsen burner or a candle. To accomplish this, hold the opposite ends of the capillary tube in your hands and place the center of the tube over the flame. When the glass starts to soften, pull the capillary tube apart in one quick motion. Gently tap the melted end onto a hard surface to remove any oddly-shaped, bent, or closed points. Note: Toothpicks can be used instead of capillary tubes; however, students often make too large of a spot with toothpicks. Flat toothpicks are preferable to thicker, rounded ones.
• Obtain hair dryers: 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 class to share with the other students.
|Table S10: Required Materials for 8 Lab Stations |Quantity |
|TLC plates |16 |
|TLC developing chambers |16 |
|Ruler |8 |
|Pencil (non-mechanical) |8 |
|Capillary tubes |56 |
|Tweezers |8 |
|Vials containing prepared solutions of adenine, thymine, and cytosine |8 each |
|Unknown Solution 1 |8 vials |
|Unknown Solution 2 |8 vials |
|Unknown Solution 3 |8 vials |
|Unknown Solution 4 |8 vials |
|Distilled water | |
|Table S11: Common Workstation |Quantity |
|Shortwave (254 nm) UV lamp |1 |
|Hair dryers |2-3 |
|Table S12: Student Workstation |Quantity |
|TLC plates |2 |
|TLC developing chambers |2 |
|Ruler |1 |
|Pencil (non-mechanical) |1 |
|Capillary tubes |7 |
|Tweezers |1 |
|Vials containing prepared solutions of adenine, thymine, and cytosine |1 each |
|Unknown Solution 1 |1 |
|Unknown Solution 2 |1 |
|Unknown Solution 3 |1 |
|Unknown Solution 4 |1 |
Procedure:
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Step 1: Prepare your TLC developing chamber(s) according to the following instructions. If there is only one chamber per group, you should run the plates one at a time in the same chamber.
To prepare your TLC developing chamber:
1. Obtain a developing chamber from the front of the room. The developing chamber consists of a beaker or plastic cup, a watch glass or piece of plastic wrap, a piece of filter paper, and, if using plastic wrap, a rubber band.
2. Place the filter paper into the chamber, as demonstrated by the image below. You may need to cut the filter paper to a smaller size to fit into the chamber. The paper should cover no more than half the surface inside the chamber. The filter paper ensures that the inside of the chamber will remain saturated with vapor from the aqueous mobile phase, so that the plate will run correctly.
[pic] [pic]
Figure S7: Examples of TLC chambers.
3. Carefully pour water into the developing chamber to a depth of approximately 0.5 cm. Water will begin to travel up the filter paper. Do not use the TLC chamber until water completely saturates the filter paper. If you wish to speed up this process, you can carefully tip the chamber to wet the filter paper.
Step 2: Prepare your TLC plates according to the directions below.
To prepare your TLC plate:
1. Using a ruler and a pencil (do not use a mechanical pencil or a pen), draw a line across each TLC plate 2 cm from the bottom, as indicated in the picture. Press the pencil lightly so as not to damage the coating on the TLC plates. This line will serve as the origin line.
2. Pressing gently, draw three hash marks across the origin line of Plate #1 (see an example hash mark in the photo below). Space these evenly across the origin line, but start and end at least 0.5 cm from the edge of the plate. Below the origin line, pressing lightly with your pencil, label hash marks with “A”, “C”, and “T” (for adenine, cytosine, and thymine). Draw four hash marks across the origin line of Plate #2. Also space these evenly across the origin line, but start and end at least 0.5 cm from the left and right sides of the plate. Below the origin line, pressing lightly with your pencil, label hash marks with “1”, “2”, “3”, and “4” (for the four unknown solutions).
[pic]
Figure S8: TLC plate with hash marks and origin line marked.
Step 3: Spot your TLC plates according to the directions below.
To spot a TLC plate:
1. You will be using capillary tubes (or toothpicks) to spot your adenine, cytosine, and thymine standards on one TLC plate and your unknown solutions on the other. Each solution will require a separate capillary tube to prevent cross-contamination. Label the top of each capillary tube with a piece of tape, using the same letters and numbers you used to label the hash marks on the TLC plate. Use caution with the capillary tubes, as they are fragile and very sharp when broken.
2. Take the capillary tube labeled “A” and place the sharpened end into your stock solution of adenine. You should be able to see the solution rise up into the tube, through capillary action.
3. Touch the end of the capillary tube gently on the origin line at the spot indicated for that solution on Plate #1. 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 tube run onto the paper. You will not use all of the solution inside the tube. If you are using toothpicks, you will spot the entire drop of adenine solution on the plate. Touch your toothpick gently to the hash mark on the origin line, being careful not to damage the surface of the TLC plate. Make sure the spot is small.
4. Repeat this process for the cytosine and thymine solutions. Also repeat for Unknown Solutions 1, 2, 3, and 4 on Plate #2.
5. Allow the TLC plate to completely dry. (You will not be able to see the spots on the origin line when it is dry.) You may blow gently on the TLC plate to speed up the drying process. If you would like to check your work, to see if you have spotted enough of your solutions onto the TLC plate, you can look at it under the shortwave UV lamp. You should see dark spots on the origin line where you placed your compounds. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step 4: Develop your TLC plates by following the procedure below. If you have two TLC chambers, you can run both plates at the same time. Just make sure you are able to pay attention to both. If you only have one TLC chamber, run Plate #1 and then run Plate #2 while Plate #1 is drying.
To develop a TLC plate:
1. Before placing your TLC plate into the developing chamber, measure and compare the height of the water in relation to the origin line on your TLC plate. If it appears that the water level of the chamber will be above your origin line when you place the 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 obtain and spot a new TLC plate. If you are unsure whether the water will cover the line, 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.
2. Without touching the plate directly with your fingers, use your tweezers to carefully place the prepared TLC plate in the developing chamber so that its bottom edge is sitting flat on the bottom of the chamber and leaning against the side of the chamber that is not covered by the filter paper. Be very careful! If the plate falls into the water, you will have to obtain and spot a new TLC plate. Make sure you record what time you first placed the plate into the chamber.
3. Cover the chamber with the plastic wrap or watch glass. If using plastic wrap, secure it with a rubber band, making sure not to move the TLC plate within the chamber. If your plate falls over, you can use your tweezers to put it back into position, provided water did not get on or above the origin line. DO NOT pick up the chamber once your plate is in place and running.
4. Watch as the mobile phase runs up the TLC plate. When the mobile phase is approximately 1 cm from the top of the plate, remove it from the chamber using your tweezers and place it face up on a paper towel. DO NOT allow the mobile phase to overrun the top of your plate.
5. Using a pencil, trace the location of the mobile phase on your plates. This is called the solvent front and is a critical step for calculation of the Rf. Note, the solvent front is sometimes not a straight line, and may be higher in the center of the plate than at the edges. This is why we make our hash marks at least 0.5 cm away from the edges of the plate.
[pic]
Figure S9: TLC plate that has been developed, indicating the position of the solvent front.
6. Once you have traced the solvent front, hold your TLC plate with your tweezers and use the blow dryer to completely dry the plate.
Step 5: Visualize the spots on your TLC plate. To do this, place your TLC plate underneath a downward facing shortwave UV lamp. Mark all spots that you see, no matter how faint, with a pencil, tracing around their outlines. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step 6: Evaluate the data for each TLC plate by following the steps in the example for each of your circled spots, and by using the equation given below:
Plate #1: The purpose of Plate #1 is to determine the Rf values of your standard solutions of nitrogenous bases (adenine, cytosine, and thymine).
Plate #2: The purpose of Plate #2 is to determine the Rf values of the unknown solutions so that you can compare them to the Rf values from Plate #1 and be able to identify the components of each unknown solution.
Calculate the retention factor (Rf) for each spot on Plate #1 and Plate #2 according to the directions below. Record these values on your data sheet. A given compound will always have the same Rf under the same conditions (e.g. stationary phase, mobile phase); this property is thus frequently used to identify unknown compounds.
Sample Rf calculation: This calculation is based on a sample that is made up of a combination of substances, and therefore has more than one spot. The procedure for determining the Rf 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.
[pic]
Figure S10: Illustration of a TLC plate including sample spots, origin line, and solvent front, to be used as an aid for calculating Rf.
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 plate out of the developing chamber. Make sure you measure from the center of each spot to the origin line to get the distance moved by the molecule.
The formula for calculating Rf:
Rf = distance moved by the molecule/solvent front
The Rf for the substance indicated by the asterisk (*) would be:
Rf = 5.5cm/6.0cm = 0.92
Adenine Synthesis in a Model Prebiotic Reaction:
Connecting Origin of Life Chemistry with Biology
Student Manual
Background Information
Billions of years ago, Earth existed only as a cloud of gas, which was so hot that even the simplest compounds could not exist. Under the influence of gravity, atoms that made up the primordial gas cloud would have begun to organize into layers, with the densest gases at the center and the least dense ones on the outer edges. As the cloud cooled, its innermost components condensed into a liquid, while the outer layers remained in the form of an envelope of gas.
Further cooling allowed for the formation of molecules: simple ones formed first, and then reacted with one another to form more complex molecules. Gradually, the Earth’s crust developed from a solid envelope of condensed compounds surrounding the hot core of the planet.
As the molten center of the Earth continued to cool, it contracted. The rigid layer above, unable to contract alongside the liquid center, formed cracks and folds through which molten rock poured out onto the surface of the planet. Modern geologic events, such as volcanic eruptions and mountain building, are evidence that these same phenomena still occur today.
At first, the Earth’s primitive atmosphere consisted of just those atoms found in the outer portion of the initial gas cloud. A combination of atmospheric cooling and volcanic activity generated an even more diverse set of molecules. While the exact composition of the Earth’s primitive atmosphere is not known, various models suggest that it contained water (H2O), carbon dioxide (CO2), methane (CH4), ammonia (NH3), and molecular nitrogen (N2). Formamide (H2NCOH) and hydrogen cyanide (HCN) were also likely present among the early molecules on Earth, and are believed to have played an important role in the chemical processes that produced the first building blocks of life, including amino acids and nucleobases.
Finally, when the Earth’s temperature fell to 100°C, it became possible for water in the atmosphere to exist in the form of liquid droplets. Continuous downpours of rain fell upon the surface of the planet, inundating it and forming an ocean. Organic molecules formed in the atmosphere dissolved into the liquid drops and accompanied them to the Earth’s surface. These compounds then encountered each other and reacted to form larger molecules, including the polymers required for the emergence of life (e.g. peptides and oligonucleotides). For additional background information on origin of life chemistry and early chemical evolution, instructors are referred to the website of the Center for Chemical Evolution () and to the book by Wills and Bada, The Spark Of Life: Darwin And The Primeval Soup (1).
Experiment 1: Adenine Synthesis in a Model Prebiotic Reaction
In this experiment, you will be using materials that were likely available on the early Earth to generate and identify a nitrogenous base of DNA and RNA using thin layer chromatography (TLC). The reactants used in this lab are formamide, ammonium formate, and diaminomaleonitrile (DAMN). The likely availability of formamide on the early Earth is supported by the discovery of this molecule in comets and other interstellar objects (2). The other reactants – ammonium formate and DAMN – can readily be generated from either formamide or hydrogen cyanide (HCN).
The reaction below illustrates how HCN can be hydrolyzed to form formamide, which can further be hydrolyzed to ammonium formate:
[pic]
Scheme S3: Chemical equilibria between HCN, formamide, and ammonium formate.
DAMN is formed from four HCN molecules in concentrated solutions of HCN (3). There are several chemical intermediates in this process, although the precise mechanism is currently unknown:
[pic]
Scheme S4: 4 molecules of HCN polymerize to form diaminomaleonitrile, or DAMN.
|Table S13: Common Workstation |Quantity |
|Short wave (254 nm) UV lamp |1 |
|Hair dryer |1 |
|Access to an oven or sand bath |1 oven or 2-3 sand baths |
|Balance |1 |
|Table S14: Student Workstation |Quantity |
|Formamide |6 mL |
|Ammonium formate |1 g |
|Diaminomaleonitrile (DAMN) |0.1 g |
|Prepared solutions of adenine, cytosine, and thymine |1 of each |
|Capillary tube/toothpick |4 |
|Test tube |1 |
|50 mL beaker |1 |
|Thermometer |1 |
|Spatula |1 |
|Weigh boat |2 |
|Test tube tongs |1 |
|Test tube rack |1 |
|Glass stirring rod |1 |
|10 mL graduated cylinder or transfer pipette |1 |
|TLC plate |1 |
|TLC developing chamber |1 |
|Ruler |1 |
|Pencil |1 |
|Tweezers or forceps |1 |
Procedure
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Step 1: Obtain and label a clean test tube with your initials.
Note: Ammonium formate, formamide, and DAMN are irritants to the skin and eyes. Wear gloves and use caution when handling these chemicals. If one of these chemicals comes in contact with your skin, wash the affected area immediately with soap and water.
Step 2: Using one of your weigh boats, measure 1.0 g 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 (or oven) for 5 minutes. Record the time on your data sheet and calculate when you will need to remove your sample; record that time on your data sheet, as well. Proceed to Step 6 while your sample is in the sand bath.
Step 6: Using your other weigh boat, measure exactly 0.1 g of the diaminomaleonitrile (DAMN) and set aside for use in Step 8.
Step 7: After 5 minutes, 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 it to the sand bath for an additional 3 minutes before moving to Step 8.
Step 8: Record the physical appearance of your solution on your data sheet. Once your test tube is cool to the touch, carefully add the DAMN you measured in Step 6 into your test tube and thoroughly mix with your stirring rod.
Step 9: Securely stopper your test tube. Place your thumb over the top of the covering on the test tube and invert your test tube several times so that all DAMN comes off 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 or oven for 20 minutes. Record the time of entry on your data sheet and calculate when you will need to remove your sample; record that time on your data sheet as well. While your test tube is in the sand bath, prepare your TLC plate and chamber. You will need room for four spots on your plate – your reaction sample and the stock solutions of adenine, cytosine, or thymine.
To prepare your TLC developing chamber:
1. Obtain a developing chamber from the front of the room. The developing chamber consists of a beaker or plastic cup, a watch glass or piece of plastic wrap, a piece of filter paper, and, if using plastic wrap, a rubber band.
2. Place the filter paper into the chamber, as shown in the image below. You may need to cut the paper to a smaller size to fit into the chamber. The paper should be taller than the TLC plate and the width of the paper should cover no more than half the surface inside the chamber. The filter paper ensures that the inside of the chamber will remain saturated with vapor from the aqueous mobile phase, so that the TLC plate will run correctly.
[pic] [pic]
Figure S11: Examples of TLC chambers.
3. Carefully pour water into the developing chamber to a depth of approximately 0.5 cm. Water will begin to travel up the filter paper. Do not use the TLC chamber until the water completely saturates the filter paper. If you wish to speed up this process, you can carefully tip the chamber to wet the filter paper.
To prepare your TLC plate:
1. Using a ruler and a pencil (do not use a mechanical pencil or a pen), draw a line across the TLC plate 2.0 cm from the bottom, as indicated in the picture below. Press the pencil lightly so as not to damage the coating on the TLC plate. This line will serve as the origin line.
2. Pressing gently, draw four hash marks across the origin line (see an example hash mark in the picture below). Space these evenly across the origin line, but start and end at least 0.5 cm from the left and right sides of the plate. Below the origin line, pressing lightly with your pencil, label hash marks with A, C, T, and R (for adenine, cytosine, thymine, and your reaction mixture).
[pic]
Figure S12: TLC plate with hash marks and origin line marked.
Step 11: After 20 minutes, use your test tube tongs to remove your sample from the sand bath or oven 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 into a small beaker. Place the remaining 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 beaker containing reaction mixture, and stir.
Step 14: Spot your TLC plate according to the directions below.
To spot a TLC plate:
1. You will be using capillary tubes (or toothpicks) to spot your adenine, cytosine, and thymine standards on the TLC plate, as well as your reaction mixture. Each solution will require a separate capillary tube to prevent cross-contamination. Label the top of each capillary tube with a piece of tape, using the same letters you used to label the hash marks on the TLC plate. Use caution with the capillary tubes, as they are fragile and very sharp when broken.
2. Take the capillary tube labeled A and insert the sharpened end into your stock solution of adenine. You should be able to see the solution rise up into the tube, through capillary action. If you are using toothpicks, dip a toothpick into the adenine solution and slowly remove it; you want a very small drop on the end.
3. Touch the end of the capillary tube gently on the origin line at the hash mark 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 tube run onto the TLC plate. You will not use all of the solution inside the tube. If you are using toothpicks, you will spot the entire drop of adenine solution on the plate. Touch your toothpick gently to the hash mark on the origin line, being careful not to damage the surface of the TLC plate. Make sure the spot is small.
4. Repeat this process for the cytosine and thymine solutions.
5. Insert the sharpened end of the final capillary tube into the beaker where you diluted your reaction product mixture with water. Spot this sample in the final location (labeled R) on your origin line. (If using toothpicks, follow the instructions in #2 and #3 above for applying samples to the toothpick and spotting on a TLC plate.)
6. Allow the spots on the TLC plate to dry completely. (You will not be able to see the spots on the origin line when they are dry.) You may blow gently on the TLC plate to speed up the drying process. If you would like to check your work, to see if you have spotted enough of your solutions onto the TLC plate, you can look at it under the shortwave UV lamp. You should see dark spots on the origin line where you placed your compounds. Record the appearance of your TLC plate on your worksheet. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step 15: Develop your TLC plate according to the directions below.
To develop a TLC plate:
1. Before placing your TLC plate into the developing chamber, measure and compare the height of the water in relation to the origin line on your TLC plate. If it appears that the water level of the chamber will be above your origin line when you place the 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 obtain and spot a new TLC plate. If you are unsure whether the water will cover the line, err on the side of caution and remove some of the water. You can always add water back with no negative effects if there is not enough water in the beaker to develop the plate.
2. Without touching the plate directly with your fingers, use your tweezers to carefully place the prepared TLC plate in the developing chamber so that its bottom edge is sitting flat on the bottom of the chamber and leaning against the side of the chamber that is not covered by the filter paper. Be very careful! If the plate falls into the water, you will have to obtain and spot a new TLC plate. Make sure you record the time when you first placed the plate into the chamber.
3. Cover the chamber with the plastic wrap or watch glass. If using plastic wrap, secure the plastic wrap over the chamber opening with a rubber band, making sure not to move the TLC plate within the chamber. If your plate falls over, you can use your tweezers to put it back into position, provided water did not get on or above the origin line. DO NOT pick up the chamber once your plate is in place and running.
4. Watch as the mobile phase runs up the TLC plate. When the mobile phase is approximately 1 cm from the top of the plate, remove it from the chamber using your tweezers and place it face up on a paper towel. DO NOT allow the mobile phase to overrun the top of your plate.
5. Using a pencil, trace the location of the mobile phase on your plate. This is called the solvent front, and marking the solvent front is a critical step for calculation of the retention factor (Rf). Note, the solvent front is sometimes not a straight line, and may be higher in the center of the plate than at the edges. This is why we make our hash marks at least 0.5 cm away from the edges of the plate.
[pic]
Figure S13: TLC plate that has been developed, indicating the position of the solvent front.
6. Once you have traced the solvent front, hold your TLC plate with your tweezers and use the blow dryer to completely dry the plate.
Step 16: Visualize the spots on your TLC plate. To do this, place your TLC plate underneath a downward facing shortwave UV lamp. Mark all spots that you see, no matter how faint, with a pencil, tracing their outlines. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step 17: Calculate the Rf of each spot according to the directions below. Use this information to identify the unknown nitrogenous base that has been synthesized by matching the Rf of your main product (largest spot) in your reaction to the Rf of one of the three standards. A given compound will always have the same Rf under the same conditions (e.g. stationary phase, mobile phase); this property is thus frequently used to identify unknown compounds.
Sample Rf calculation: The calculation below is for a single compound (spot) in a sample that is a mixture of substances, and therefore produces more than one spot on the TLC plate. The procedure for determining the Rf for each spot within one sample is exactly the same as the procedure for determining the Rf value for many individual samples on one plate.
[pic]
Figure S14: Illustration of a TLC plate including sample spots, origin line, and solvent front, to be used as an aid for calculating Rf.
The solvent front is the distance the mobile phase traveled on the plate, and is what you recorded by a pencil line when you took your TLC plate out of the developing chamber. Make sure you measure from the center of each spot to the origin line to obtain the distance moved by the molecule.
The formula for calculating Rf is:
Rf = distance moved by the molecule / distance moved by the solvent front
The Rf for the substance indicated by the asterisk (*) would be:
Rf = 5.5 cm/6.0 cm = 0.92
Experiment 2: Extraction of DNA from Strawberries
Background Information
In today’s experiment, you will break apart the cells of a strawberry and isolate the DNA (deoxyribonucleic acid) in a form that is visible to the naked eye. Wild strawberries are diploid, meaning they contain two copies of each chromosome. Commercially produced strawberries are octoploid. Because they contain eight copies of each chromosome, they are particularly good to use for this lab.
The DNA in today’s experiment will precipitate, or come out of solution, as long white strands. The white material is actually thousands, or even millions, of DNA strands (and associated proteins) wrapped around each other. An individual strand of DNA is so small that it can only be imaged using the most specialized microscopes. The “images” that helped Watson and Crick to uncover the double helix structure of DNA were actually diffraction patterns acquired by Rosalind Franklin, who passed X-rays through DNA fibers, much like the fibers of DNA that you will isolate today.
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 step is accomplished, in part, by the physical act of smashing the strawberries in a Zip-top bag. Ripened fruit is also used in this lab because the cell walls are already weakened during 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 the membranes while leaving the DNA intact.
The third step is the separation of the DNA from the coarse, 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 and therefore, not visible in the filtered strawberry mixture. However, DNA is insoluble in ethanol in the presence of salt. Thus, the careful addition of ethanol onto the top of the DNA mixture will cause the DNA to come out of solution and form visible, insoluble threads that can be spooled and collected.
|Table S15: Student Workstation |Quantity |
|Strawberry |1 |
|Zip-top bag |1 |
|DNA extraction buffer |25 mL |
|250 or 100 mL beaker |1 |
|Additional 100 mL beaker |1 |
|Cheesecloth/coffee filter/Kimwipe |5 |
|Test tubes |2 |
|Test tube stopper/Parafilm/aluminum foil |1 |
|10 mL graduated cylinder |1 |
|Ice-cold ethanol |50 mL |
|Stirring rod |1 mL |
|Transfer pipette |1 or 2 |
Procedure
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Step 1: Remove the green stem from one strawberry and place the strawberry into a plastic zip-top bag.
Step 2: Remove the air from the bag, seal it, and mash the strawberry for two minutes, 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 one minute or until the solution is thoroughly mixed with the 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 the 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 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 the 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 the DNA precipitate out of solution as an opaque white substance at the interface between the ethanol and the filtered solution. Occasionally, the DNA may precipitate as a clear substance, which can be seen because of bubbles formed, but it will still be at the interface of the ethanol and the filtered solution. Make a sketch of what you see and record your observations of the precipitated DNA on your data sheet.
Step 11: Allow the DNA solution to sit undisturbed for five minutes. Remove the DNA that has accumulated at the surface by spooling it with a stirring rod or transfer pipette and place it into a test tube. If using a transfer pipette, move as little of the ethanol as possible when transferring the DNA.
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 will be DNA.
Step 13: Fill a 1 mL transfer pipette with water, and add about 0.5 mL 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 to your sample without your teacher’s approval. Record the amount of water used on your data sheet.
Note: Dissolving the DNA should cause the solution to become thicker and cloudier, and most of the white strands of the DNA will disappear.
Step 15: Place a stopper into the test tube, label your test tube, and place it in the designated location.
Experiment 3: Hydrolysis of DNA
Background Information
The DNA within all living organisms, from bacteria to humans, is made up of the exact same chemical components: deoxyribose, phosphate, and four nitrogenous bases: adenine (A), thymine (T), cytosine (C), and guanine (G). The difference in the amount of DNA and the specific arrangement of the nitrogenous bases (i.e. the genetic code) is what accounts for the wide variation of life forms on Earth. In very similar organisms, such as in humans and other primates, the information encoded by DNA is almost identical. Comparing the similarities between the DNA sequences (i.e. genomes) of two species, or two individuals of the same species, can help scientists determine the relatedness of two species or individuals.
The primary structure of DNA is generally described in terms of nucleotides, the molecular unit defined as a nucleobase connected by a glycosidic bond at the 1′ position of a phosphorylated 2′-deoxyribose sugar (Figure S15). Along the backbone of a DNA polymer, or strand, nucleosides are connected to each other through the phosphate groups. A phosphodiester linkage exists between the 3′ position of each nucleoside and the 5′ position of the next nucleoside along a given DNA strand (Figure S15). The order of the nucleotide bases (A, G, C and T) along a strand is the chemical basis for coding the genetic information contained within a DNA polymer. The repeating 3′-phosphate-5′ linkages between nucleotides provide the polymer directionality for DNA.
In eukaryotic cells, DNA is contained within the nucleus and the mitochondria, as well as in the chloroplast of photosynthetic organisms. In bacteria, DNA is contained within a less well-defined structure known as the nucleoid that, unlike the eukaryotic nucleus, is not surrounded by a membrane. The secondary structure of DNA within living organisms is predominantly a double helix formed by two strands with complementary nucleoside sequences, that is, sequences that support Watson-Crick base pairing along their entire lengths (i.e. A paired with T; G paired with C). The two strands of a Watson-Crick double helix run in opposite directions (as defined by the directionality of their polymer backbones) (Figure S15). Notice how the sugar molecules in Figure S15 below are oriented differently on the opposite sides of the paired bases. A Watson-Crick duplex is therefore said to have an antiparallel arrangement of its two strands. This twisted structure of two base-paired DNA polymers is the reason that DNA is commonly referred to as a double helix.
[pic][pic]
Figure S15: DNA Structure. (Left) The chemical structure of DNA. The bond indicated by a line between the nitrogen atom of adenine (A) and the 1´ carbon of the deoxyribose sugar is the bond that is broken during the depurination reaction that releases adenine from the DNA backbone. (Right) A schematic representation of the DNA double helix emerging from a chromosome. Right image courtesy of Genome Management Information System, Oak Ridge National Laboratory. .
The four nucleobases of DNA, adenine (A), thymine (T), cytosine (C), and guanine (G), are of two chemical classes: the purines (A and G) and the pyrimidines (T and C) (Figure S16). As shown above in Figure S15, in a DNA duplex, each base pair is formed by the hydrogen bonding of a purine nucleobase with a pyrimidine nucleobase. That is, adenine forms a base pair with thymine and guanine forms a base pair with cytosine.
[pic]
Figure S16: The chemical structures of the nucleobases of DNA.
Hydrogen bonds are not only important for the formation of Watson-Crick base pairs, but are also the bonds that hold together liquid water and are the bonds used within living cells to guide a multitude of molecular interactions. Thus, it is important to appreciate the nature of hydrogen bonds. 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 a single water molecule. In the case of water, the oxygen is more electronegative, or has a higher attraction for the shared electrons than does the 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 and a partially negative oxygen or nitrogen atom in neighboring molecules. Hydrogen bonds involving both oxygen and nitrogen atoms are responsible for base pairing in DNA (Figure S15).
In today’s experiment, you will be using the DNA extracted in the previous laboratory, and you will treat it with heat and acid to release one of its nitrogenous bases. Since the nitrogenous bases all have slightly different chemical structures, the bond that attaches them to the deoxyribose of the backbone varies in strength. The process of breaking apart a macromolecule like DNA into its monomers and smaller building blocks is called hydrolysis, and requires the use of water. The breaking of a bond by hydrolysis can be seen as the opposite of the formation of the same bond, which results in the production of a water molecule. Bonds that can be hydrolyzed include those that hold together the amino acids in proteins (the peptide bonds) and the nucleotides of DNA (the nucleobase-deoxyribose glycosidic bonds, and the phosphate-deoxyribose phosphate-ester bonds). Such bonds are often called reversible bonds, which allow living organisms to recycle the building blocks of proteins and nucleic acids.
To prepare the isolated genomic DNA for partial hydrolysis (that is, release of a nucleobase), the DNA is first dissociated from proteins, called histones, which package the DNA into its chromosomal structure. Separating DNA from its bound histones is accomplished by heating. Secondly, a strong acid, such as the HCl used in this experiment, is required. 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 proton, to the purine bases, which catalyzes the cleavage of the bond that holds these nucleobases to the sugar-phosphate backbone. After partially hydrolyzing the DNA, you will use thin layer chromatography (TLC) to determine the identity of the base that has been liberated from the polymer by cleavage of its bond with deoxyribose (the glycosidic bond).
|Table S16: Common Workstation |Quantity |
|Access to a 95°C – 100°C hot water bath |1 or 2 |
|Access to a hair dryer |1 or 2 |
|Access to a short wave (254 nm) UV lamp |1 |
|Table S17: Student Workstation |Quantity |
|DNA solution prepared in DNA extraction lab | |
|Test tube |1 |
|Pair of test tube tongs |1 |
|Test tube rack |1 |
|Rubber stopper |1 |
|TLC plate |1 |
|Capillary tubes/toothpicks |4 |
|Ruler |1 |
|Pencil (non-mechanical) |1 |
|Pair of tweezers or forceps |1 |
|TLC chamber |1 |
|10 mL graduated cylinder (or 3 transfer pipettes) |1 |
|3M HCl |4 mL |
|Distilled water | |
|Thermometer |1 |
|500 mL beaker |1 |
|25 mL beaker |1 |
|Hot plate |1 |
Procedure:
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Part 1
Step 1: Obtain a 500 mL beaker and a thermometer. Fill the beaker with 350 mL of water and place it onto the hot plate. Turn the hot plate 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 2 mL of your extracted DNA solution and place it into your labeled test tube.
Step 4: Cover the test tube. Check the temperature of your hot water bath. When it 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.
Note: Hot plates may vary. If your water bath begins boiling, turn it down slightly. If, after some time, it has not reached 95°C, you may need to turn the temperature up.
Step 5: Heat the sample for 30 minutes, 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 minutes for 30 minutes.
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.
Step 7: Allow your test tube to cool to the touch. Carefully add 2 mL of water and 4 mL of 3M HCl to your DNA solution. NOTE: HCl is a strong acid. Use caution when handling this substance, and wash your skin immediately with water for several minutes if it spills on you.
Step 8: Cover your test tube and place it back into the 95°C hot water bath. Record the time. Measure the temperature every 10 minutes and record that information on your data sheet.
Step 9: After 30 minutes, use your test tube tongs to remove your test tube from the water bath. Put it in the storage location designated by your teacher. If your class period will be over in less than 30 minutes, your teacher will complete this step for you.
Teacher’s note: For a 45-60 minute class, you may need to remove students’ test tubes from the water bath after the HCl treatment. For a block schedule, students will be able to do this step themselves.
Part 2
Step 1: Obtain your hydrolyzed DNA solution from the previous laboratory period. Record the appearance of your DNA solution after heating and treatment with HCl.
Step 2: Prepare your TLC plate and chamber. If you do not remember how to do this, please refer to the instructions from the “Adenine Synthesis” laboratory. You will be running four samples on your TLC plate – your hydrolyzed DNA and the adenine, cytosine, and thymine standard solutions you used in the “Synthesis of Adenine” lab.
Step 3: 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 4: Spot the adenine, cytosine, and thymine standard solutions onto your plate at the appropriate locations. Make sure your spots are completely dry, and then run your TLC plate in the chamber. Refer to the instructions from the “Adenine Synthesis” laboratory if you do not remember how to do this step.
Step 5: When the mobile phase is approximately 1 cm from the top of the plate, remove it from the chamber and immediately trace the location of the solvent front with a pencil.
Step 6: Dry your plate using a hair dryer, and use the UV lamp 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 the light facing down at all times.
Step 7: Calculate the Rf of your sample and of the adenine, cytosine, and thymine standards. Identify the nitrogenous base liberated from your DNA sample. If you do not remember how to calculate Rf, please refer to the “Adenine Synthesis” experiment.
Experiment 4 (Optional): Thin Layer Chromatography
Background Information
Chromatography is a method of separating and identifying chemicals in mixtures containing two or more compounds. The separation is accomplished by the distribution of the components of the mixture between two phases: one that is stationary and one that moves, or is mobile. A variety of solid stationary phases exist, and the mobile phase can be liquid or gas. Different compounds will have different solubilities and interactions with the stationary and mobile phases; chromatography takes advantage of these properties to allow for the separation of complex mixtures of compounds.
Thin layer chromatography, or TLC, is a technique in which the stationary phase is solid and the mobile phase is liquid. The stationary phase that you will use in today’s laboratory is silica gel that covers and is supported by a plastic backing. Alumina is another common stationary phase used in TLC. The mobile phase you will use is water; depending on the compounds you wish to separate by TLC, many other mobile phases are available.
TLC is commonly used to monitor whether organic reactions have proceeded to completion and to test purification conditions for synthesized organic compounds. It is also a useful technique for learning about a mixture of unknowns – for example, determining the number components present in a mixture and their identities.
The different distance each molecule travels along the adsorbent (stationary phase) in relation to how far the mobile phase has traveled is called the retention factor (Rf) and can be used to identify an unknown.
This laboratory is designed to build your TLC skills. In the other laboratories of this four-part series, you will use TLC to identify a nitrogenous base that you have synthesized in a model prebiotic reaction, and a nitrogenous base that you have hydrolyzed from strawberry DNA.
|Table S18: Common Workstation |Quantity |
|Shortwave (254 nm) UV lamp |1 |
|Hair dryers |2-3 |
|Table S19: Student Workstation |Quantity |
|TLC plates |2 |
|TLC developing chambers |2 |
|Ruler |1 |
|Pencil (non-mechanical) |1 |
|Capillary tubes |7 |
|Tweezers |1 |
|Vials containing prepared solutions of adenine, thymine, and cytosine |1 each |
|Unknown Solution 1 |1 |
|Unknown Solution 2 |1 |
|Unknown Solution 3 |1 |
|Unknown Solution 4 |1 |
Procedure:
READ ALL INSTRUCTIONS BEFORE BEGINNING EACH STEP.
Step 1: Prepare your TLC developing chamber(s) according to the following instructions. If there is only one chamber per group, you should run the plates one at a time in the same chamber.
To prepare your TLC developing chamber:
1. Obtain a developing chamber from the front of the room. The developing chamber consists of a beaker or plastic cup, a watch glass or piece of plastic wrap, a piece of filter paper, and, if using plastic wrap, a rubber band.
2. Place the filter paper into the chamber, as demonstrated by the image below. You may need to cut the filter paper to a smaller size to fit into the chamber. The paper should cover no more than half the surface inside the chamber. The filter paper ensures that the inside of the chamber will remain saturated with vapor from the aqueous mobile phase, so that the plate will run correctly.
[pic] [pic]
Figure S17: Examples of TLC chambers.
3. Carefully pour water into the developing chamber to a depth of approximately 0.5 cm. Water will begin to travel up the filter paper. Do not use the TLC chamber until water completely saturates the filter paper. If you wish to speed up this process, you can carefully tip the chamber to wet the filter paper.
Step 2: Prepare your TLC plates according to the directions below.
To prepare your TLC plate:
4. Using a ruler and a pencil (do not use a mechanical pencil or a pen), draw a line across each TLC plate 2 cm from the bottom, as indicated in the picture. Press the pencil lightly so as not to damage the coating on the TLC plates. This line will serve as the origin line.
5. Pressing gently, draw three hash marks across the origin line of Plate #1 (see an example hash mark in the photo below). Space these evenly across the origin line, but start and end at least 0.5 cm from the edge of the plate. Below the origin line, pressing lightly with your pencil, label hash marks with “A”, “C”, and “T” (for adenine, cytosine, and thymine). Draw four hash marks across the origin line of Plate #2. Also space these evenly across the origin line, but start and end at least 0.5 cm from the left and right sides of the plate. Below the origin line, pressing lightly with your pencil, label hash marks with “1”, “2”, “3”, and “4” (for the four unknown solutions).
[pic]
Figure S18: TLC plate with hash marks and origin line marked.
Step 3: Spot your TLC plates according to the directions below.
To spot a TLC plate:
1. You will be using capillary tubes (or toothpicks) to spot your adenine, cytosine, and thymine standards on one TLC plate and your unknown solutions on the other. Each solution will require a separate capillary tube to prevent cross-contamination. Label the top of each capillary tube with a piece of tape, using the same letters and numbers you used to label the hash marks on the TLC plate. Use caution with the capillary tubes, as they are fragile and very sharp when broken.
2. Take the capillary tube labeled “A” and place the sharpened end into your stock solution of adenine. You should be able to see the solution rise up into the tube, through capillary action.
3. Touch the end of the capillary tube gently on the origin line at the spot indicated for that solution on Plate #1. 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 tube run onto the paper. You will not use all of the solution inside the tube. If you are using toothpicks, you will spot the entire drop of adenine solution on the plate. Touch your toothpick gently to the hash mark on the origin line, being careful not to damage the surface of the TLC plate. Make sure the spot is small.
4. Repeat this process for the cytosine and thymine solutions. Also repeat for Unknown Solutions 1, 2, 3, and 4 on Plate #2.
5. Allow the TLC plate to completely dry. (You will not be able to see the spots on the origin line when it is dry.) You may blow gently on the TLC plate to speed up the drying process. If you would like to check your work, to see if you have spotted enough of your solutions onto the TLC plate, you can look at it under the shortwave UV lamp. You should see dark spots on the origin line where you placed your compounds. Record the appearance of your TLC plate on your worksheet. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step 4: Develop your TLC plates by following the procedure below. If you have two TLC chambers, you can run both plates at the same time. Just make sure you are able to pay attention to both. If you only have one TLC chamber, run Plate #1 and then run Plate #2 while Plate #1 is drying.
To develop a TLC plate:
1. Before placing your TLC plate into the developing chamber, measure and compare the height of the water in relation to the origin line on your TLC plate. If it appears that the water level of the chamber will be above your origin line when you place the 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 obtain and spot a new TLC plate. If you are unsure whether the water will cover the line, 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.
2. Without touching the plate directly with your fingers, use your tweezers to carefully place the prepared TLC plate in the developing chamber so that its bottom edge is sitting flat on the bottom of the chamber and leaning against the side of the chamber that is not covered by the filter paper. Be very careful! If the plate falls into the water, you will have to obtain and spot a new TLC plate. Make sure you record what time you first placed the plate into the chamber.
3. Cover the chamber with the plastic wrap or watch glass. If using plastic wrap, secure it with a rubber band, making sure not to move the TLC plate within the chamber. If your plate falls over, you can use your tweezers to put it back into position, provided water did not get on or above the origin line. DO NOT pick up the chamber once your plate is in place and running.
4. Watch as the mobile phase runs up the TLC plate. When the mobile phase is approximately 1 cm from the top of the plate, remove it from the chamber using your tweezers and place it face up on a paper towel. DO NOT allow the mobile phase to overrun the top of your plate.
5. Using a pencil, trace the location of the mobile phase on your plates. This is called the solvent front and is a critical step for calculation of the Rf. Note, the solvent front is sometimes not a straight line, and may be higher in the center of the plate than at the edges. This is why we make our hash marks at least 0.5 cm away from the edges of the plate.
[pic]
Figure S19: TLC plate that has been developed, indicating the position of the solvent front.
6. Once you have traced the solvent front, hold your TLC plate with your tweezers and use the blow dryer to completely dry the plate.
Step 5: Visualize the spots on your TLC plate. To do this, place your TLC plate underneath a downward facing shortwave UV lamp. Mark all spots that you see, no matter how faint, with a pencil, tracing around their outlines. Warning: UV light is damaging to both your eyes and your skin. Make sure that you are wearing your safety goggles. Do not look directly into the UV lamp and keep it facing downwards at all times.
Step Six: Evaluate the data for each TLC plate by following the steps in the example for each of your circled spots, and by using the equation given below:
Plate #1: The purpose of Plate #1 is to determine the Rf values of your standard solutions of nitrogenous bases (adenine, cytosine, and thymine).
Plate #2: The purpose of Plate #2 is to determine the Rf values of the unknown solutions so that you can compare them to the Rf values from Plate #1 and be able to identify the components of each unknown solution.
Calculate the retention factor (Rf) for each spot on Plate #1 and Plate #2 according to the directions below. Record these values on your data sheet. A given compound will always have the same Rf under the same conditions (e.g. stationary phase, mobile phase); this property is thus frequently used to identify unknown compounds.
Sample Rf calculation: This calculation is based on a sample that is made up of a combination of substances, and therefore has more than one spot. The procedure for determining the Rf 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.
[pic]
Figure S20: Illustration of a TLC plate including sample spots, origin line, and solvent front, to be used as an aid for calculating Rf.
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 plate out of the developing chamber. Make sure you measure from the center of each spot to the origin line to get the distance moved by the molecule.
The formula for calculating Rf:
Rf = distance moved by the molecule/solvent front
The Rf for the substance indicated by the asterisk (*) would be:
Rf = 5.5cm/6.0cm = 0.92
Appendix A: Student Worksheets
Included on the following pages are sample student worksheets for each experiment. Feel free to edit them as appropriate for your classes’ needs. Each begins on a new page to facilitate printing and copying.
Name: _______________________________ Period: ___________ Date: ____________
Adenine Synthesis in a Model Prebiotic Reaction
Mass of ammonium formate: ___________
Volume of formamide: ___________
Mass of DAMN: ___________
Temperature of oven/sand bath in °C: ___________
Temperature of oven/sand bath in °F: ___________
Work space for temperature conversions
°C = (5/9)* (°F-32)
°F = (9/5)* °C+32
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 |Description of physical appearance |
|4 | |
|8 | |
|9 | |
|11 | |
Sketch your TLC plate, before and after development:
Before After
|Substance |Calculations |Rf |
|Adenine | | |
|Cytosine | | |
|Thymine | | |
|Reaction Mixture | | |
Identity of nitrogenous base synthesized in the DAMN reaction: ________________
Post-Laboratory Questions:
Please answer these questions on a separate sheet of paper.
1. Why did the densest gases sink to the center of the enormous, hot cloud that was the primordial Earth, instead of sinking to the bottom of the cloud?
2. List the four nitrogenous bases (also called nucleobases) that are found in DNA. Indicate which bases are purines and which are pyrimidines.
3. Did you think it would be this easy to synthesize an essential component of life? Write down your thoughts on the significance of making 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 Section.
6. Identify the number of sigma and pi bonds in formamide (refer to the Background Section for the structure).
7. How do you think HCN was formed on the early Earth? (Hint: see Background Section regarding the suspected molecules in the atmosphere of the early Earth.)
8. There are two different functional groups in the DAMN molecule that contain nitrogen. How many lone pairs of electrons are on each of them?
Name: _______________________________ Period: ___________ Date: ____________
Extraction of DNA from Strawberries
Objective of DNA Extraction Experiment:______________________________________
________________________________________________________________________
________________________________________________________________________
Volume of filtered strawberry solution: __________
Volume of ethanol needed (3X the amount of strawberry solution): __________
Sketch of DNA solution/precipitate:
Observations about DNA Solution: ___________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Volume of water used to dissolve the DNA: __________
Post-Laboratory Questions:
Please answer these questions on a separate sheet of paper.
1. Commonly cultivated bananas are triploid and wheat is usually hexaploid. How many copies of each chromosome do these organisms have?
2. In many species of bees, wasps, and ants, males develop from an unfertilized egg, and are monoploid as a result. How many copies of each chromosome do 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 this is an important step?
4. In the background section, four major steps were listed. Identify each of these, and determine which steps in the protocol they match with.
5. Construct a concept map using at least ten of the bold words found in the background section.
Name: _______________________________ Period: ___________ Date: ____________
Hydrolysis of DNA
Objective of DNA hydrolysis experiment:______________________________________
________________________________________________________________________
________________________________________________________________________
Part 1:
Time in water bath: ________
Time out of water bath: ________
Temperature of water bath:
0 minutes: ________
10 minutes: ________
20 minutes: ________
30 minutes: ________
Observations of DNA solution in Part 1:
Part 2:
Time in water bath: ________
Time out of water bath: ________
Temperature of water bath:
0 minutes: ________
10 minutes: ________
20 minutes: ________
30 minutes: ________
Observations of DNA solution in Part 2:
Sketch your TLC plate, before and after development:
Before After
Refer to the “Adenine Synthesis” laboratory for the equation to calculate Rf, if you need to refresh your memory.
|Substance |Calculations |Rf |
|Adenine | | |
|Cytosine | | |
|Thymine | | |
|Unknown Base from Hydrolyzed DNA | | |
Identity of unknown nitrogenous base: _________________
Post-Laboratory Questions:
Please answer these questions on a separate sheet of paper.
1. The definition of chemical hydrolysis is given in the background information. 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. Construct a paragraph using at least 10 of the words in bold type.
Additional questions (for chemistry classes):
5. Give three examples of strong acids.
6. Describe how the arrangement of electrons leads to the formation of partial charges.
7. Explain the difference between the attraction of partial charges to one another and the attraction of ions.
Name: _______________________________ Period: ___________ Date: ____________
Thin Layer Chromatography (TLC)
|Sample |Distance Traveled |Location of Solvent Front |Rf |
|Adenine standard | | | |
|Cytosine standard | | | |
|Thymine standard | | | |
|Unknown 1 | | | |
|Unknown 2 | | | |
|Unknown 3 | | | |
|Unknown 4 | | | |
Sketch your TLC plates below:
Plate 1 Plate 2
Identify the nitrogenous bases in the unknown solutions:
Unknown 1: ___________________________
Unknown 2: ___________________________
Unknown 3: ___________________________
Unknown 4: ___________________________
Justify your answers using your results. Evidence may be quantitative or qualitative in nature. Be sure to use the correct terminology.
Post-Laboratory Questions:
Please answer these questions on a separate sheet of paper.
1. What applications might TLC have in other areas of science? Give two specific examples.
2. What were potential sources for error in this experiment?
Appendix B: Teacher Answer Guides
Experiment 1: Adenine Synthesis in a Model Prebiotic Reaction
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?
The densest gases sank to the center of the primordial Earth because gravity is a central force.
2. List the four nitrogenous bases (also known as nucleobases) that are found in DNA. Indicate which bases are purines and which are pyrimidines.
Purines: adenine and guanine
Pyrimidines: cytosine and thymine
3. Did you think it would be this easy to synthesize an essential component of life? Write down your thoughts on the significance of making a nitrogenous base in the classroom.
Any answer is acceptable.
4. Which nitrogenous base did you hypothesize would be generated? Did your hypothesis match your result?
Answers will vary.
Additional Questions for Chemistry Classes:
5. Identify and draw at least one functional group found in a molecule from the background section.
[pic]
Figure S21: Functional groups.
1: nitrile or cyano group
2: amino group
3: aldehyde group
4: amide group
5: carboxyl group or carboxylic acid
Note: R can be a carbon or a proton in each case. A carboxylic acid can also be drawn in its ionized form with a negative charge on the single-bonded oxygen.
6. Identify the number of sigma and pi bonds in formamide (refer to the background section for the structure).
5 sigma bonds: 1 for each single bond in the molecule, and 1 for the double bond
1 pi bond: 1 for the double bond between carbon and oxygen
7. How do you think HCN was formed on the early Earth?
HCN was formed through interactions among substances on the early Earth and/or on other objects in the solar system such as comets or meteors. More specifically, HCN could have formed as the result of energy, such as electric discharges and UV light, causing reactions in an atmosphere containing methane, ammonia, and hydrogen. This possibility is supported by the early Miller experiments, where HCN was found to be produced when a methane, ammonia, hydrogen, and water vapor atmosphere was subjected to spark discharges (for further information, see Miller, S.L., Production of Some Organic Compounds under Possible Primitive Earth Conditions, J. Am. Chem. Soc. 77, 2351-2361, 1955).
8. There are two different functional groups in the DAMN molecule that contain nitrogen. How many lone pairs of electrons are on each of them?
1 lone pair on amino nitrogens
1 lone pair on cyano nitrogens
Experiment 2: Extraction of DNA from Strawberries
1. Commonly cultivated bananas are triploid and wheat is usually hexaploid. How many of each type of chromosome do these organisms have?
Triploid organisms contain three copies of each chromosome, and hexaploid organisms contain six copies of each chromosome.
2. In many 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 they have? What is a more common name for monoploid?
Monoploid organisms have one copy of each chromosome; these organisms are also called haploid.
3. In Step 9, you are instructed to pour your ethanol down the side of the beaker. Why do you think this is an important step?
To prevent the ethanol and water from mixing, thereby creating two separate layers with a clean interface where the DNA will precipitate.
4. In the Background Section, four major steps were listed. Identify each of these, and determine which steps in the protocol they match with.
1: Break apart the cell wall (procedure Steps 1 and 2).
2: Disrupt the cellular and nuclear membranes to release the DNA (procedure Step 3).
3: Filter DNA from excess strawberry material (procedure Steps 4, 5, and 6).
4: Precipitate DNA (procedure Steps 8 and 9).
5. Construct a concept map using at least ten of the bold words found in the Background Section.
Student answers will vary. Number of words used can be increased for advanced classes or used as an extra credit opportunity.
Experiment 3: Hydrolysis of DNA
1. The definition of chemical hydrolysis is given in the background information. What do you think enzymatic hydrolysis means?
Enzymatic hydrolysis of DNA is the breaking down of DNA by enzymes (DNases), where the enzymes catalyze the addition of a water molecule across the bond that is being broken.
2. Is DNA soluble in water? How do you know?
DNA is soluble in water, because of the “like dissolves like” principle – both substances are polar. Also, this property was observed in the DNA extraction laboratory.
3. Draw a picture that illustrates the term “anti-parallel.”
Any picture illustrating anti-parallel strands of DNA is acceptable.
4. Construct a paragraph using at least 10 of the words in bold type.
Student answers will vary.
Additional questions (for chemistry classes):
5. Give three examples of strong acids.
Hydrochloric acid (HCl), hydrobromic acid (HBr), nitric acid (HNO3), sulfuric acid (H2SO4), hydroiodic acid (HI), perchloric acid (HClO4), etc.
6. Describe how the arrangement of electrons leads to the formation of partial charges.
The more electronegative element in a polar bond attracts the electrons towards its side, creating a partial negative charge. The other atom in the bond therefore gains a partial positive charge with this loss of electron density.
7. Explain the difference between the attraction of partial charges to one another and the attraction of ions.
The attraction between partial charges is temporary and weak. Ions, however, are permanently positively and negatively charged, with a full unit charge (or more) on each, and therefore the attraction between them is stronger.
Experiment 4: Thin Layer Chromatography
1. What applications might TLC have in other areas of science? Give two specific examples.
Answers may vary but should generally relate to separating the components of mixtures, detection of contaminants, checking the progress of a synthesis reaction, etc.
2. What were potential sources for error in this experiment?
Answers will vary but may include spotting too much or too little on the TLC plate, the TLC plate falling over into the water, too much or too little water in the TLC chamber, the filter paper touching the TLC plate, running the TLC plate for a time that is too short or too long, contamination of standard solutions or capillary tubes, etc.
Appendix C: Safety Information
|Table S20: CAS Numbers and Detailed Safety Information |
|Chemical |CAS Registry Number |Notes |
|Adenine |73-24-5 |Follow general lab safety practices. |
|Cytosine |71-30-7 |Follow general lab safety practices. |
|Thymine |65-71-4 |Follow general lab safety practices. |
|Formamide |75-12-7 |Irritant when inhaled or ingested. Wear gloves, if |
| | |possible, when handling this chemical. Wash thoroughly with|
| | |soap and water if formamide contacts the skin. If inhaled, |
| | |remove to fresh air. (This chemical is not volatile except |
| | |at elevated temperatures.) |
|Ammonium Formate |540-69-2 |Irritant to the eyes and skin. Wear gloves when handling |
| | |this chemical. Wash thoroughly with soap and water if |
| | |ammonium formate contacts the skin. Toxic to mucous |
| | |membranes. |
|Diaminomaleonitrile |1187-42-4 |May be harmful if swallowed, inhaled, or absorbed through |
| | |skin. Wear gloves when handling this chemical. Wash |
| | |thoroughly with soap and water if diaminomaleonitrile |
| | |contacts the skin. |
|Sodium Chloride |7647-14-5 |Follow general lab safety practices. |
|Ethanol |64-17-5 |Causes severe eye and respiratory system irritation. |
| | |Flammable liquid. |
|Hydrochloric Acid |7647-01-0 |Corrosive and incompatible with metals. In concentrated |
| | |form, causes severe damage to skin and eyes. Inhalation of |
| | |vapor is extremely harmful. Ingestion may be fatal. Handle|
| | |with extreme caution. |
Notes:
• If disposable gloves are not available for students, it may be preferable for the teacher to perform the formamide + ammonium formate + DAMN reaction as a demonstration and provide the reaction product to students to spot on TLC plates.
• Waste from the synthesis of adenine lab must be deposited into an appropriate organic waste container and procedures for hazardous waste disposal, which may vary by school, must be followed.
• Waste from the DNA extraction lab may be poured down the sink. Waste from the DNA hydrolysis lab must be neutralized (e.g. with baking soda) before disposal. After neutralization, it may also be poured down the sink.
• The water from TLC developing chambers may be poured down the sink.
• Several steps in these labs involve the heating of samples. Please be cautious with hot objects and/or samples and handle only with test tube tongs or heat-resistant gloves.
• If using capillary tubes, please be aware that the ends may be sharp, and handle with caution. Do not dispose of glass capillary tubes in the trash as this may cause injury to maintenance or cleaning staff.
• Plastic-backed TLC plates may be disposed of in the trash. Do not dispose of glass-backed TLC plates or glass vials in the trash, but instead place them into an appropriate broken glass container.
• Shortwave UV light is used to visualize spots on the TLC plates. UV light is harmful to the skin and eyes. Students should not look directly into the UV lamp, and should keep it facing downward at all times.
Appendix D: Glossary
Adenine: C5H5N5, a purine which is found in DNA, where it base pairs with thymine, and in RNA, where it base pairs with uracil; adenine is also found in many cofactors that are required for enzymes to function properly (as in ATP)
Adsorbent: a substance that is attracted to a surface and hence collects on the surface in a layer; an adsorbent may be a dissolved solid, a gas, or a liquid
Ammonium formate: composed of an ammonium ion (NH4+) and deprotonated formic acid (HCO2-), it is a colorless solid; this compound can be formed by the addition of a water molecule to formamide
Chromatography: a physical method of separating mixtures containing two or more components; these components are carried in one direction in a mobile phase (liquid or gas) through a stationary phase (a column or surface containing specific chemical functional groups) and are separated based on their differential interactions with the stationary and mobile phases
Cytosine: C4H5N3O, a pyrimidine that is found in DNA and RNA, where it base pairs with guanine
Deoxyribonucleic acid (DNA): a nucleic acid that stores hereditary information in all living organisms
Diaminomaleonitrile: also known as DAMN or HCN tetramer, an organic compound with the formula C4H4N4 that is believed to be a precursor to adenine in prebiotic reactions; contains two nitrile groups and two amino groups
Electrostatic force: the attraction or repulsion between two particles based on their charges or partial charges
Eukaryotic cell: an organism such as yeast, algae, or the cells of any multicellular organism that contains membrane-bound organelles and a nucleus
Formamide: a polar solvent and clear liquid which is miscible with water; a plausibly prebiotic compound with the formula CH3NO that, when heated in the presence of minerals, gives rise to a variety of compounds that may have been important to early life
Hydrogen bonding: the sharing of a hydrogen atom by two electronegative atoms, only one of which is covalently bonded to it
Hydrolysis: reaction of a compound with water; this may lead to bond cleavage and decomposition, as in the hydrolysis of DNA, or the formation of another compound, as in the hydrolysis of HCN to form formamide
Ionizes: when an atom or molecule takes on a positive or negative charge via the addition or loss of a proton or a change in oxidation state (as in that of a metal)
Mixture: consists of two or more substances (in any proportion) which may be separated by exploiting their physical properties (such as in distillation, filtration, and chromatography)
Monomer: the smallest unit of a polymer; monomers of nucleic acids are called nucleotides
Nonpolar molecule: a molecule that is symmetrical (such as nitrogen gas, N2 or methane, CH4) and uncharged, or in which there are not substantial differences in the electronegativity between the atoms
Nucleotide: the basic unit of RNA or DNA; contains a base, a sugar, and a phosphate group
Octoploid: having eight sets of chromosomes
Partial charge: when electrons are shared unequally by the two molecules in a polar bond, the slight positive or negative character of the atoms in the bond (less than a full unit of charge)
Polar molecule: a molecule in which the electrons are shared unequally between atoms and partial charges do not cancel out in space (as in water, which has a bent structure)
Precipitate: a solid that comes out of solution as the result of a chemical reaction, the mixture of solutions containing incompatible components (such as salt water and ethanol, with DNA as the precipitate), or centrifugation of a suspension
Protonation: the addition of a proton to an atom or molecule; this occurs in the presence of an acid, or proton donor
Purine: a class of organic compounds containing five carbons and four nitrogens (at the 1, 3, 7, and 9 positions) of fused five- and six-membered rings; purine derivatives include adenine and guanine as well as caffeine and uric acid
Pyrimidine: a class of organic compounds containing four carbons and two nitrogens (at the 1 and 3 positions) in one six-membered ring; pyrimidine derivatives include cytosine, thymine, and uracil
Retention factor (Rf): a unitless number between 0 and 1 that describes how far a solute travels on the stationary phase in thin layer chromatography (TLC); when standard solutions are run on the same TLC plate as a mixture of unknowns, the retention factor can be used to identify unknown compounds
Solubility: how much of a compound that can be dissolved in another compound to form a homogeneous solution
Solvent front: in thin layer chromatography (or paper chromatography), the distance the mobile phase has traveled at any given time; can be visualized as the edge between the wet and dry sections of a TLC plate
Thin layer chromatography (TLC): a physical separation technique using a solid stationary phase (a plate backed with aluminum, plastic, or glass and coated with silica, alumina, or another substance) and a liquid mobile phase, which may be water, an organic solvent, or a mixture of liquids
Thymine: C5H6N2O2, a pyrimidine found in DNA, which base pairs with adenine
X-ray crystallography: a method for determining the structure of molecules based on the location of electron density in the molecules
Appendix E: References
1. Wills, C.; Bada, J. L. The Spark Of Life: Darwin And The Primeval Soup; Basic Books: New York, 2000.
2. Rubin, R. H.; G. W. Swenson, J.; Solomon, R. C.; Flygare, H. L. Astrophys. J. 1971, L39-L44.
3. Joyce, G. Nature 1989, 338, 217-224.
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