CP Biology ’12-13 Name______
Date______Period_____
Lab: Isolation of Deoxyribonucleic Acid
Objective: The purpose of this lab is to treat cells (either human cheek cells or banana cells) with a variety of materials in order to break open the cell membrane and the nuclear envelope, releasing the 46 chromosomes from each cell into solution. Many thousands of cells will be used for the extraction, so that you will be combining the DNA from many nuclei. You will be able to see long, combined strands of DNA.
DNA is ______
DNA is found in all living organisms, it is a common thread which ties life together. Bacteria, blue-green algae, yeast, worms and humans all share the same genetic or hereditary molecule—DNA.
Recent studies indicate that not only do we share this molecule with all other living things, but some of the genes within our DNA are the same as those found in other organisms.
A gene is a 1) ______of DNA that codes for a
2) ______or ______
3) It is a unit of ______
This similarity certainly makes sense when you consider that, for example, most organisms undergo aerobic cellular respiration to make ATP. Each of those organisms will need the same enzymes for the biochemical reactions which make up cellular respiration so each will need the same gene to code for those enzymes. Just think, your cells contain some of the same genes as your pet hamster, dog—or iguana!
Recombinant DNA Technology:
Today, scientists can locate genes that code for important human proteins and cut them out of the chromosome where they are located. These genes can then be placed inside another organism such as a simple prokaryotic bacteria transforming it into a human protein factory! Insulin, the protein hormone needed to provide your cells with glucose for cellular respiration, is one such protein.
In the past, the only source of this insulin for diabetics was from other mammals such as pigs and cows. Unfortunately, this insulin was slightly different than human insulin and it caused problems for some people. Today, we can ‘cut’ the insulin producing gene from human DNA and insert it into a bacteria called E. coli. The bacteria then produces human insulin, called Humulin, which can be given to diabetics.
Introduction to the laboratory procedure:
Many different procedures can be used to help isolate the DNA from cells. Our lab isn’t as advanced as those used in DNA Technology but we do have the ability to isolate DNA form the other chemicals in a cell. The following are different methods and their purpose in DNA isolation.
· Salt: Adding salt to a slurry of cells will help to break down the cell membrane by denaturing the proteins in the membrane. Remember that salt (NaCl) is an ionic compound that dissociates in an aqueous solution to form Na+ and Cl-. The individual ions will interact with and disrupt hydrogen bonds and other R-group interactions between amino acids. Initially this helps to break down the cell membrane and the nuclear membrane. The proteins associated with the DNA in chromatin also need to be denatured, so that purified DNA can be extracted. Additionally, proteins dissolved in the cytoplasm will be denatured.
· Liquid detergent: The detergent will act to dissolve or separate the lipid components of the cell membrane, and to give access to the proteins and nucleic acids within the cell. Detergent molecules are similar in structure to phospholipids. They have both hydrophobic and hydrophilic regions. The similar regions of both detergent and phospholipids interact with each other, disrupting the structure of the cell membrane and causing it to break apart. Household detergent acts in a similar way when it breaks apart the lipid molecules in dirt and grease, so they can more easily be washed away when cleaning dishes or clothes.
· Cold, 95% ethanol: DNA is soluble in water but not very soluble in alcohol (and even less so when the alcohol is cold), and it will begin to precipitate or separate out of the solution so it an easily be seen as a whitish cloud in the cold alcohol layer.
You will be treating the cells with salt and liquid detergent in order to isolate the DNA. The material you will get will be white, slimy and gathered from thousands of cells. Although you won’t be able to put it under the microscope to observe the double helix (it is much too small to see) you will be able to collect and hold DNA!
You have the option of choosing to isolate DNA from your own cheek cells OR from banana cells. Plant cells have ten times the amount of DNA compared to human cells and make excellent subjects for DNA isolation. The human genome contains approximately 3.5 Billion base pairs. Plant cells contain 35 billion base pairs! Which do you choose – banana or cheek cells?______
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OPTION I: ISOLATING DNA FROM HUMAN CHEEK CELLS
Materials for Isolating Human Cheek Cell DNA:
· Plastic test tube· 3 ml plastic pipettes with suction bulb
· 8% sodium chloride solution
· beaker to hold test tube
· Gatorade, graduated cylinders
· bathroom drinking cup / · 10 % sodium lauryl sulfate solution (dish detergent)
· plastic wrap
· small, plastic pipette
· Alcohol (95% ethyl or isopropyl)-chilled
· toothpick
· 1-3 dram, screw-top vial
Directions for Isolation of HUMAN CHEEK CELL DNA: All the materials used in this experiment can be purchased at a grocery store. This is something you CAN do at home! The directions for this lab are extremely easy. Use the following procedure to ensure success.
1) Add 0.8 ml sodium chloride solution to the plastic test tube. Set the tube in a beaker.
2) Using a graduated cylinder, pour 6 ml of Gatorade into a clean bathroom cup.
3) Pour the Gatorade into your mouth. Swirl the Gatorade around for at least two minutes. The more active the swirling, the better. You can also bite on the inside of your cheek to get more cells.
4) Spit the Gatorade back into the drinking cup.
(PROCEDURE CONTINUED ON NEXT PAGE)…
RELEASE THE DNA FROM INSIDE THE CHEEK CELLS:
5) Pour the cheek cell solution into the test tube that contains the salt solution.
6) Add 0.8 ml of the 10% sodium lauryl sulfate solution to the mixture in the test tube.
7) Place plastic wrap over the top of the test tube and mix the contents by GENTLY INVERTING THE TEST TUBE. Do not shake the test tube!
8) Draw off any bubbles from the top of the test tube with a small, plastic pipette. Discard the pipette!
PRECIPITATE THE HUMAN CHEEK CELL DNA:
9) Hold the test tube at a slight angle, carefully add alcohol down the side of the tube so it forms a layer over the cheek cell mixture.
10) Place the test tube in the beaker to sit for a few minutes. Observe what happens at the interface between the ethyl alcohol and the cheek solution. The clouds of white strands you are observing are the DNA!
COLLECT THE HUMAN CHEEK CELL DNA:
11) Carefully use a clean toothpick to collect the DNA. Transfer some of it to one of the small, alcohol filled vials for storage. Cap your DNA tightly and it should last a long time.
SAFETY CLEANUP: Toss anything that came in contact with your mouth or the cheek solution into the trash!
OPTION II: ISOLATING DNA FROM BANANA CELLS
Materials for Isolation of Banana Cell DNA:
· ½ a banana· 2 clear, plastic cups
· small, white cup with 5 g of salt
· 5 ml Palmolive dish detergent
· graduated cylinder
· plastic spoon and knife / · cheesecloth
· glass rod with twisted end (use with care!)
· flasks of alcohol and small vials for DNA storage
· rubbing alcohol on ice
· water baths at 50-55oC
Directions for Isolating Banana Cell DNA: All the materials used in this experiment can be purchased at a grocery store. This is something you CAN do at home! The directions for this lab are extremely easy. Use the following procedure to ensure success.
Partner I / Partner IIStep I. Obtain ½ of a banana and place it in the clear, plastic cup. Use the plastic knife to cut it into small pieces. / Step I. Use the weigh paper at the balance station to weigh out 5 grams of salt. Place the salt in the white cup and return it to your worktable.
Step II. Add 5 grams of salt to the banana. / Step II. Use the graduated cylinder to measure 14 ml of tap water. Give this to your partner.
Step III. Using the spoon, mash the banana pieces. Then add 14 ml of water. Keep mashing until the mixture is free of any lumps and has the same consistency as baby food.
(CONTINUED ON NEXT PAGE)… / Step III. Prepare the cheesecloth or other material and fold it so the banana mixture can be poured out onto the cloth.
Step IV. Spoon the mixture onto the center of the cloth. Get the second, clear plastic cup ready for your partner. Measure 5 ml of dish detergent into the graduated cylinder. Wash the plastic spoon. / Step IV. Gather the sides of the cloth, and squeeze the cloth so that all the liquid from the mixture drips into the second clear, plastic cup.
Step V. Empty the detergent into the liquid that your partner has squeezed into the cup. Gently stir with the clean spoon. Try not to create bubbles while stirring. / Step V. Obtain chilled alcohol and pour carefully down the inside of the plastic cup. The alcohol will sit on top or the banana liquid. Stop pouring when you have equal-sized layers of banana liquid and alcohol.
Step VI. Wait 5 minutes.
Step VII. You should notice a cloudy white precipitate forming at the interface or point of contact between the banana mixture and the alcohol. You are looking at DNA!
Step VIII. Carefully use the glass rod to collect the DNA. Transfer some of it to one of the small, alcohol filled vials for storage. Cap your DNA tightly and it should last a long time.
CP Biology ’12-13 Name ______Date______Period: ______
REVIEW QUESTIONS – DNA ISOLATION LAB
Questions: Use information from your notes packet and the this lab to answer the questions. Do not copy the information directly from either packet – rather, put it into your own words.
1) What was the source of DNA in this lab? ______
2) Approximately how much DNA is in the sample you extracted? (example: 1 strand, 46 strands, thousands of strands, etc.) Explain your answer. ______
3) What was the role of the salt in this experiment? Explain how it works. ______
4) In addition to salt, heat can be used to help disrupt the cells. Go back to your Biochemistry notes and explain in detail, on a molecular basis, how heating the solution would break down the cells. ______
5) What was the role of the detergent in this experiment?
6) What was the purpose of pouring the cold alcohol over the cell solution? In your answer, explain what it means for something to be soluble or insoluble.
7) Phenol red is a pH indicator dye that changes from red to yellow in an acidic solution. If you add phenol red to the DNA collected at the end of the experiment, it will turn yellow. Why? Explain your answer.
8) Scientists often have a hard time deciding on a test subject for their investigation because any organism has its “pro’s” and “con’s”. Defend your choice of cells (banana or human cheek cells) in this investigation.
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