Module 2 · Web viewModule 2 DNA Files Investigation 2.1 What is DNA? Biotechnology – Extract DNA from Cells (Recursos en Español) DNA Gel Electrophoresis Simulation DNA Fingerprint

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

Module 2DNA Files

Investigation 2.1 What is DNA? Activity 1: Biotechnology

How Extract DNA from Cells

Objectives1. 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 tubesfunnelfilter paperglass rods (wooden sticks)cauliflower, dry peas, strawberries ( or other fruit), liver (beef or chicken), sweetbread (beef thymus gland), wheat germ, cheek cells.Mortar and pestleblenderplastic baggies

ProcedureCheek 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 30-45 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.

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.

Activity 2: DNA Gel Electrophoresis Simulation

Materialsfood coloring (represent DNA samples)mix equal parts of blue and redmix equal parts of blue, red and yellow (the primary colors)beakers (or plastic containers)

foilfilter paper (cut in rectangles)rubbing alcoholwaterrulersQ-tips

Other samples: leaves, berries, flowers, black felt pen, KoolAid –different flavors, candy (Skittles or M&M’s)

Procedure1. 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.

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.

2. What color dye traveled the fastest? _____________________

3. What color dye traveled the slowest? _____________________

4. What is DNA gel electrophoresis? _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Activity 3: DNA Fingerprint and ForensicsWho ate the Apple?

Procedure1. 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 ate the apple? ___________________________________

Who is Guilty?

Adam

Ali

Addl

eyAa

ron

Arno

ld

Anto

nioAl

lisha

Allen

Akee

mAl

lison

Armo

nd

Abiga

il

Ashl

ey

Activity 4: Build a DNA Molecule

MaterialsGenetic Science Learning Center - handoutlicorice (red and various colors)marshmallows (gummy bears)toothpicksDNA paper template – Adapted from Access Excellence handoutcolor pencilsscissors

ProcedureOption 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- orange2. Color and cut out paper nucleotide templates.3. Students can work in groups4. Use tape to assemble DNA molecule

Taylor, Tish . “Discovering DNA Structure.” Access Excellence. 1994 Woodrow Wilson Biology Institute. http://www.accessexcellence.org/AE/AEPC/WWC/1994/discovering_dna.html

Activity 5: Central DogmaDNA – RNA - Protein

MaterialsDNA ACTIVITY CARDs: TAC, ATC, etc. Print template cards and laminate.

Activity 5: Proteins and ProteomicsCentral Dogma

DNA----------RNA-------------- ProteinMaterialsGenetic Science Learning Center - handoutlicorice (red and various colors)marshmallows (gummy bears)round shapes (or Post-It paper)toothpicksscissors

ProcedureOption 1:

1. Follow procedure in the Genetics Science Learning CenterReading DNA http://learn.genetics.utah.edu/units/basics/print-and-go/reading_DNA.cfm

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?

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.

Procedure1. 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 #1TAC AGG TCA GGC GTC AAA TTG ATC CCG ATT

Protein_________________________________________________

DNA sequence #2TCA GTC TAC TAT GGC TAG CCT AAT GCG CCG ATC AAA

Protein_________________________________________________

DNA sequence #3TTA TAC GGC ATG TTA GGC CGC CCC GGA TTA GGT ACT TTA

Protein_________________________________________________

Alternative Splicing Student WorksheetName ____________________________________________________

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.

21 3 4 5 6

Procedure1. Squares 1-6 represent exons. Exons are the parts of a gene that

become translated into proteins. Use coloring pencils to color the squares 1-6.

2. 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

1 3 6

1

m-RNA 1

m-RNA 2

2 4 5 6

m-RNA 3

2 3 5 6

Investigation 2.2 DNA Gel Electrophoresis

Lab procedures adapted with permission from General

Biology for Non-majors Lab Manual, Phillips, M., McGraw- Hill, 2009.

PurposeTo introduce students to the theory and methods used to perform agarose gel electrophoresis. What is electrophoresis? Electrophoresis uses electricity to separate molecules according to size. It is one of the most important procedures used in studying DNA.

Case ScenarioScenario adapted from BIOTECH Project, University of Arizona, “Mystery of the Stolen Cat Food.” http://biotech.bio5.org/content/activities#DNAFingerprinting

Mystery of the Stolen Cat Food

Fluffy is my white haired cat. She sometimes likes to have her meals on the back porch, so I put her dish of cat food outside. Recently, she’s been whining and complaining because she’s hungry. After doing a little detective work, I realized that another white haired cat has been eating Fluffy’s food. However, there are 2 other white haired cats in my neighborhood and I can’t tell which cat is eating Fluffy’s food. You are going to help me figure out which cat is the criminal!

MaterialsBioRad electrophoresis equipment and food color dyes. DNA Electrophoresis

1. Balance2. Small weigh boat and scoop3. Power supply4. Plastic electrophoresis box with tray 5. Comb6. Masking tape7. Micropipette8. Box of tips9. 1 large beaker for discarding used tips.10. 250 mLflask11. 50 mL graduated cylinder12. Rulers13. Agarose (BioRad)14. TAE Buffer (BioRad)15. Practice gels and practice loading dye (loading dye – ½ glycerin,

½ water, 1-3 drops of blue food coloring).

16. Crime scene food color samples (DNA), mixed with glycerin. Prepare samples ahead of class.

One sample of each food color: ½ glycerin, ½ water, 1-3 drops of food coloring: red, yellow, blue, green, and mystery mix.Tube #1 - saliva from food (mystery mix)Tube #2 - Fluffy (red)Tube #3 - white cat #1 (yellow)Tube #4 - white cat #2 (blue)Tube #5 - white cat #3 (mystery mix)

Making an agarose gelPrepare a 1.0% agaorse gel: Weigh 0.4 g of agarose and place it in a 250 mL flask with 40 mL of 1X TAE buffer solution. Note: you can prepare agarose gel of 0.8% by placing 0.32 g of agaorse and place it in a flask with 40 mL of TAE buffer.

Note gels may be prepared ahead of time. Cover flask with saran wrap and heat in microwave for one minute

(agarose should boil) Use gloves to remove flask Swirl gently to be sure all of the agarose is dissolved. Wait until it cools enough so it feels warm to the touch but will not

burn. (temp of baby’s milk bottle).

Preparing Gel Tray Prepare Gel Tray: Use masking tape to tape each end of the gel tray. Be sure the tape fits tightly. You may want to tape twice around. Insert comb. Pour dissolved agarose into the tray, avoid creating bubbles. If any bubbles appear, use a plastic pipette to move them to the side and out

of the way. Wait approximately 20-30 minutes until the gel sets (appears opaque).

Be careful not to move or jar gel while it is solidifying. Rinse the flask immediately with warm tap water then rinse flask 3

times with distilled water. Swirl around each time.

How to Use a Micropipette PracticePractice micropipetting: Your instructor will demonstrate the proper use of the micropipette.

While the gel is solidifying, practice micropipetting 10 µL of practice loading dye. Remember, you are using very small amounts; 1 L is equal to 1 millionth of a Liter. See the 1 L flask for comparison.

ALWAYS put a plastic tip on the micropipette. Push the pipette into the plastic tip.

Hold the micropipette so your thumb is on top of the dispensing knob or plunger. (shake hands with the pipette).

ALWAYS keep micropipette vertically (up) when there is fluid in it. Never allow fluid to run back into the pipette by laying it down filled.

Use your thumb to control the speed of the plunger. ALWAYS release (lift your thumb up) SLOWLY.

The micropipette has two stops. Press to the first stop. Push down harder to the second stop. SLOWLY release (lift your thumb up).

Hold the micropipette in one hand vertically about eye level and bring your sample tube to the same level.

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).

Filling the sample well: Use the sample provided to practice loading a sample gel.

To fill the sample well: 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.

Eject the used tip in a beaker. ALWAYS change tips for each new sample you need to pipette.

Loading DNA Sample1. Gently remove tape and comb from gel tray and place the tray in the

electrophoresis box. Be sure the wells are at the negative end. The DNA samples will run to the positive end.

2. Measure 250 mL of buffer (1x TAE). Pour prepared running buffer solution (approximately 250 mL) into the gel box reservoirs to cover the gel. The gel should be completely covered about 1 mm in depth. Do not overload. Be sure no air is trapped underneath the gel tray.

Loading samples3. Press to the first stop. Insert the pipette tip just under the surface of

the DNA sample. SLOWLY, release (let your thumb up).Fill your well by pressing down to the first stop. Then, continue to press to the second stop to release all the fluid. DO NOT RELEASE until you remove the tip from the well, then SLOWLY release.

Electrophoresis4. Avoid shock by making sure the table top is dry. Do not turn the power

on until it is dry. 5. Connect the top of the box with voltage cables. The negative cable is

black and the positive cable is red. Be sure the wells are at the negative end.

6. Plug power supply into the outlet. Turn on Power. Turn voltage to 100 volts and run for approximately 25-30 minutes. Look through the apparatus to see migration.

7. Turn off Power supply. 8. Disconnect leads from power supply.

Student Name____________________________________________

DNA Electrophoresis Student Worksheet1. What is electrophoresis? __________________________________________________________________________________________________________________

2. Use color pencils to draw the gel results.

3. Which cat ate Fluffy’s food? __________________________

Websites/ReferencesDNA Extraction Virtual and Electrophoresis Lab Learn Genetics University of Utah http://learn.genetics.utah.edu/units/biotech/index.cfmBiotech Project-The Univerisity of Arizona http://biotech.bio5.org/content/activities#DNAFingerprintingHuman Genome Project Information – Image Gallery http://www.ornl.gov/sci/techresources/Human_Genome/education/images.shtmlDNA Replication image – scroll down to image (many images available)http://genomics.energy.gov/gallery/basic_genomics/gallery-01.htmlFingerprint E.colihttp://reconstructors.rice.edu/extra/cdc/Cracking the Code of Life – go to Sequence Yourselfhttp://www.pbs.org/wgbh/nova/genome/Putting DNA to Work -Science Museum of the National Academy of Scienceshttp://www.koshland-science-museum.org/exhibitdna/index.jspProteomics and Cancer Fact sheethttp://www.cancer.gov/cancertopics/factsheet/proteomicsqaAccess Excellence http://www.accessexcellence.org/DNA Interactive. Cold Springs Harbor laboratory 2003. 10 April 2007.

http://www.dnai.org/Learn.Genetics. The Biotechniques Virtual Laboratory. University of Utah

2007. 10 April 2007.

http://learn.genetics.utah.edu/units/biotech/index.cfmTaylor, Tish . “Discovering DNA Structure.” Access Excellence. 1994

Woodrow Wilson Biology Institute. http://www.accessexcellence.org/AE/AEPC/WWC/1994/discovering_dna.html