Module 2. DNA Files. Investigation 2.1 What is DNA? Biotechnology Extract DNA from Cells (Recursos en

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1 Module 2 DNA Files Investigation 2.1 What is DNA? Biotechnology Extract DNA from Cells (Recursos en Español) DNA Gel Electrophoresis Simulation DNA Fingerprint and Forensics Who Ate the Apple? Build a DNA Molecule (Recursos en Español) Proteins to Proteomics (Recursos en Español) Investigation 2.2 DNA Gel Electrophoresis Making an Agarose Gel How to Use a Micropipette Loading and Electrophoresis Staining and Understanding Gel Results Investigation 2.3 PCR (Polymerase Chain Reaction)

2 Module 2 DNA Files Investigation 2.1 What is DNA? Activity 1: Biotechnology How Extract DNA from Cells Objectives 1. Students will learn how to extract DNA from plant and animal cells. Materials salt solution (6g of salt to 94 ml of water) soap solution (1 part soap to 3 parts water) Note: You can combine the soap and salt solution to make buffer solution. If you want students to experiment with what is the best method to extract DNA from cells, having separate bottles of salt and soap works best. 91% alcohol (keep alcohol in the freezer and on ice during the experiment). Alcohol can be purchased in drug stores. test tubes funnel filter paper glass rods (wooden sticks) cauliflower, dry peas, strawberries ( or other

3 fruit), liver (beef or chicken), sweetbread (beef thymus gland), wheat germ, cheek cells. Mortar and pestle blender plastic baggies Procedure Cheek Cells 1. Pour approximately a teaspoon of water into a small Dixie cup. If you have too much water, empty the cup. 2. Swish the water in your mouth vigorously for at least seconds. 3. Place cell/ water mixture back into your Dixie cup. 4. Pour cell/water mixture to fill a test tube to approximately one inch full. 5. Add 40 drops of soap solution and 40 drops of salt solution to your test tube and gently mix with your wooden stick. Try not to create soap bubbles! 6. Add cold alcohol down the sides of your test tube until you have twice the amount of the cell/buffer mixture. 7. DNA will precipitate (come out of solution) and look like white mucous. 8. Use your wooden stick or glass rod and twirl as you remove (loop) the DNA. Procedure Beef/chicken liver and sweet bread (thymus gland) 1. Cut a piece of liver (2 x 2 ). You do not need very much. 2. Add approximately 40 drops of salt and 40 drops of soap solution to your mortar and pestle. Note you may add more soap solution if it is too dry. 3. Grind the mixture. 4. Use a funnel lined with filter paper and a test tube to filter your cell/buffer mixture. Try not to break the filter paper as you squeeze. If it is too dry add more soap solution.

4 5. Add cold alcohol down the sides of your test tube until you have twice the amount of the cell/buffer mixture. 6. DNA will precipitate (come out of solution) and look like white mucous. 7. Use your wooden stick or glass rod and twirl as you remove the DNA. Procedure Inquiry-based 1. Allow students to experiment with the DNA Extraction procedures. Students can change the concentration of salt and/or soap solution. 2. Students can collaborate to see the procedure that yields the most DNA extraction. Instructor s Notes: DNA is located on chromosomes. There are 46 chromosomes in each of our cells. Half (23 chromosomes) come from the father and half (23 chromosomes) come from the mother. Chromosomes have banding patterns of dark and light regions. The dark bands show regions where genes are located. Only 2% of the DNA has genes (approximately 20,000-25,000 genes). Genes are the basic unit of heredity and contain information to make proteins. The rest (98%) of the DNA may help to regulate when and how much proteins to make. Proteins control many of the cell s functions and make up most of the cell s structure. Proteins regulate many of the cell s activities.

5 Activity 2: DNA Gel Electrophoresis Simulation Materials food coloring (represent DNA samples) mix equal parts of blue and red mix equal parts of blue, red and yellow (the primary colors) beakers (or plastic containers) foil filter paper (cut in rectangles) rubbing alcohol water rulers Q-tips Other samples: leaves, berries, flowers, black felt pen, KoolAid different flavors, candy (Skittles or M&M s) Procedure 1. Draw a line across the bottom of 2 pieces of filter paper approximately 2 centimeters from the edge. 2. Use a different toothpick for each sample. 3. Make a spot (evenly spaced) of each sample across the bottom line of your filter paper. 4. Place one of filter papers in alcohol and one in water. 5. Be sure the pencil line stays above the liquid. NOTE: The solvent will separate the dyes present according to their molecular size and give distinct banding patterns. The smaller size molecules in the dye will travel farther and faster than the larger sized molecules. This is like what happens in DNA gel electrophoresis. Instead of using solvents to separate the different size molecules, we use electricity! 6. When the liquid reaches the top (approximately 1 ½ centimeters from the top), remove the filter paper and draw a line to indicate how far the molecules traveled. 7. You can repeat the procedure using other samples (leaves, berries, Skittles, black felt pen.

6 DNA Gel Electrophoresis Simulation Student Worksheet Name Tape your filter paper here 1. Compare the banding patterns present on your filter paper from both the alcohol and water solvents. Do you see any differences in the banding patterns, sizes, colors? Explain what you see.

7 2. What color dye traveled the fastest? 3. What color dye traveled the slowest? 4. What is DNA gel electrophoresis?

8 Activity 3: DNA Fingerprint and Forensics Who ate the Apple? Procedure 1. Handout Who is Guilty? 2. Transparency template for each student. Problem: Someone ate Mrs. Phillips apple. The hungry thief left behind saliva on the apple core. DNA saliva samples of the possible suspects have been fingerprinted using DNA gel electrophoresis. 3. Match the known DNA saliva sample the hungry thief- to see who ate the apple. Who is Guilty? Adam Ali Addley Aaron Arnold Antonio Allisha Armond Akeem Allison Allen Abigail Ashley Who ate the apple?

9 Activity 4: Build a DNA Molecule Materials Genetic Science Learning Center - handout licorice (red and various colors) marshmallows (gummy bears) toothpicks DNA paper template Adapted from Access Excellence handout color pencils scissors Procedure Option 1: Make a model of DNA using licorice and marshmallows (or gummy bears). Option 2: 1. Give each students a nucleotide template to color as follow: Phosphate red Sugar (leave white) Adenine blue Thymine green Guanine- yellow Cytosine- orange 2. Color and cut out paper nucleotide templates. 3. Students can work in groups 4. Use tape to assemble DNA molecule

10 Taylor, Tish. Discovering DNA Structure. Access Excellence Woodrow Wilson Biology Institute.

11 Activity 5: Central Dogma DNA RNA - Protein Materials

12 Activity 5: Proteins and Proteomics Central Dogma DNA RNA Protein Materials Genetic Science Learning Center - handout licorice (red and various colors) marshmallows (gummy bears) round shapes (or Post-It paper) toothpicks scissors Procedure Option 1: 1. Follow procedure in the Genetics Science Learning Center Reading DNA Option 2: 1. Print amino acid cards (or write amino acids on 3X5 card). You may need to write an amino acid several times depending on the protein you build. 2. Print DNA, m-rna, and t-rna cards. 3. Distribute cards so that each student has a card. 4. TAC is the START codon. ACT is the STOP codon. Have students with the DNA cards line up (or place on the table top or floor) between the start and stop codons. 5. Students with the m-rna cards position themselves (or place on the table top or floor) complimentary to the DNA. 6. t-rna cards pick up the correct amino acids and position themselves along the m-rna. 7. Demonstrate alternative splicing. Remove one or two of the exons in the m-rna message. Now, what proteins are made?

13 What is Proteomics? Proteomics is the study of all the proteins in an organism. There are approximately 20,000 30,000 genes that code for over 100,000 proteins in the human body. How are proteins encoded, how do they interact with one another, what function do they serve in biological pathways, how are proteins expressed in various cells and at different times in the life cycle of organisms are just of the few questions scientists are studying. Procedure 1. Handout amino acid codon Genetics Learning -Utah. 2. Use the following DNA sequences to assemble amino acids. You can abbreviate the amino acid. Remember the start and stop codons! DNA sequence #1 TAC AGG TCA GGC GTC AAA TTG ATC CCG ATT Protein DNA sequence #2 TCA GTC TAC TAT GGC TAG CCT AAT GCG CCG ATC AAA Protein DNA sequence #3 TTA TAC GGC ATG TTA GGC CGC CCC GGA TTA GGT ACT TTA Protein

14 Alternative Splicing Student Worksheet Name How can approximately 25,000 genes in our DNA code for over 100,000 proteins? One answer is alternative splicing. More than one protein can be made from one gene. Procedure 1. Squares 1-6 represent exons. Exons are the parts of a gene that become translated into proteins. Use coloring pencils to color the squares Using your color pencils, make the following proteins. alternative splicing m-rna 1 - exon 1, 3, 6 alternative splicing m-rna 2 exon 1, 2, 4, 5, 6 alternative splicing m-rna 3 exon 2, 3, 5, 6 m-rna exons m-rna m-rna m-rna

15 Investigation 2.2 DNA Gel Electrophoresis Materials Handout of sample gel result Electrophoresis equipment and prepared DNA samples. Activity 1: Making an agarose gel 1. Weigh 0.2g of agarose and place it in a flask with 25 ml of the buffer solution. 2. You can microwave for one minute or cover with aluminum foil and boil until agarose is completely dissolved. 3. Use gloves to remove flask from the hot plate and let it cool. It should cool enough so that it feels warm to the touch. 4. Add one drop of dye. 5. Prepare the gel tray by placing the rubber dams (thick part on top) at each end of the tray. Place the comb in the gel tray. 6. Be sure the comb is not touching the bottom of the gel tray. 7. Gently pour the melted agarose into the tray. Make sure you do not create bubbles. If you have any bubbles, take a pipette and move them out of the way. 8. Let the gel harden for minutes. Activity 2: How to Use a Micropipette 1. Hold the micropipette as if you were shaking hands with it. 2. Always put a plastic tip. Be sure it is on securely. 3. The micropipette has two stops. Press down until you feel a resistance. This is the first stop. Push down harder to the second stop. SLOWLY release (lift your thumb up). 4. Press to the first stop and do not release. Insert the pipette tip just under the surface of the solution. SLOWLY, release (let your thumb up). 5. Practice using the micropipette. 6. Use the sample provided to practice loading a sample gel.

16 Activity 3: Loading and Running Gel Electrophoresis 1. Press to the first stop and continue to press to the second stop to fill the well with sample fluid. DO NOT RELEASE until you remove the tip from the well. SLOWLY release. If you do not keep plunger down until you remove the tip from the well, you will suck liquid back into the tip. 2. Eject the used tip in a beaker. 3. ALWAYS change tips for each new sample you need to pipette. 4. Remove the comb and rubber dams from the gel tray. 5. Place the tray in the electrophoresis box. 6. Be sure the wells are at the negative end (black terminal). 7. Measure 250 ml of buffer and add 2 drops of dye. 8. Pour buffer solution into the electrophoresis box. The buffer will fill both compartments and cover the gel approximately 2 mm. 9. Do not overfill and check to be sure no bubbles are trapped underneath the tray. 10. Be sure the work area is dry. 11. Connect the terminals (positive and negative) to the power supply. Set the voltage to 140 volts. 12. Run electrophoresis for minutes or until the lead dye is approximately ¾ across the gel. 13. Turn off the power supply and unplug it. Remove terminals from the power supply. Activity 4: Staining and understanding gel results 1. Remove the tray from the gel box. 2. Carefully place the gel in a large weighing boat (or plastic container) and cover it with stain for approximately 20 minutes. Gently shake the gel a few times throughout the 20 minute staining time. 3. Pour the stain into the designated container. 4. Pour enough distilled water to cover the gel. You can leave overnight or for minutes to remove excess stain,

17 DNA Gel Sample

18 What is DNA? Student Worksheet Name 1. The sides (uprights) of the DNA molecule are made up of alternating and molecules. 2. The base adenine (A) bonds with the base. 3. The base cytosine (C) bonds with the base. 4. A nucleotide is made up of 3 parts:, and 5. Why is soap and salt added to the DNA cell mixture? 6. Why is alcohol added to the cell mixture? 7. What does the DNA look like when it is spooled? 8. DNA has an overall negative charge. Draw an arrow under each well to indicate the movement of the DNA sample in an electrical field. _ What are the approximate sizes of the two DNA fragments in lane 2? and 11. What is the approximate size of the DNA samples in lane 4 and 5? and 12. T or F. The smaller DNA fragments are seen closer to the well.

19 Websites/References DNA Extraction Virtual and Electrophoresis Lab Learn Genetics University of Utah Human Genome Project Information Image Gallery html DNA Replication image scroll down to image (many images available) Fingerprint E.coli Cracking the Code of Life go to Sequence Yourself Putting DNA to Work -Science Museum of the National Academy of Sciences Proteomics and Cancer Fact sheet Access Excellence DNA Interactive. Cold Springs Harbor laboratory April Learn.Genetics. The Biotechniques Virtual Laboratory. University of Utah April Taylor, Tish. Discovering DNA Structure. Access Excellence Woodrow Wilson Biology Institute. dna.html

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