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Cheek cell DNA extraction lab - Biology Isolation of DNA from

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					Bio H 10          Isolation of DNA from Human Cheek Cells                   Name ______________________________

Objective: The purpose of this lab is to treat human cheek cells with a variety of materials in order to break
open the plasma membrane and the nuclear envelope, releasing the 46 chromosomes from each cell into
solution. The DNA can then be isolated from the solution to be used in many ways including genetic studies,
disease identification and DNA fingerprinting.
You will see how easy the extraction is by using simple chemicals and procedures that could be done at home.
Many thousands of cheek cells will be used for the extraction, so that you will be combining the DNA from
many cells. You will be able to visually see clumps of DNA molecules obtained from your own cheek cells.
Introduction:
Different procedures can be used to help isolate the DNA from cells. During isolation, the membranes holding
the nuclear DNA are broken and the DNA is separated from proteins associated with the molecule. We will use
salt, detergent and alcohol to break open the cell and separate the DNA from the proteins that surround it. Heat
can also be used but we will not be using it in our method.
     Salt: Adding salt to 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 in the proteins imbedded in the membrane. This disrupts the shape of
         the proteins in both the plasma membrane and the nuclear membrane. These membranes become
         unstable and break open, releasing the cytoplasm and the DNA from the nucleus of the cell. In addition,
         proteins that are associated with the DNA, like histones, are denatured and separated from the DNA.
     Heat (55oC): High temperatures, like the addition of salt, will also help denature proteins in the cell.
         Care must be taken to keep the temperature below 60oC so that the DNA is not denatured. Temperatures
         above 60oC will start to denature the DNA by breaking hydrogen bonds that connect the double strands.
     Liquid detergent: The detergent will act as an emulsifier
         to dissolve or separate the phospholipid components of
         the plasma membrane, and to give access to the nucleic
         acids within the cell. Detergent molecules are similar in
         structure to bile salts. 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                      Phospholipids
                                                                                          and some
         it to break apart. Household detergent acts in a similar                        proteins are
                                                                                            inside
         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). DNA molecules have very low density
         and will float to the top of an aqueous solution like the cheek cell solution you will be preparing.
         Alcohol also has a lower density than water. When the alcohol is poured on top of the solution with
         DNA, the DNA will move up into the alcohol. DNA does not dissolve as readily in the alcohol as it
         does in the cheek cell solution so it will precipitate out or clump together to form a whitish cloud. This
         mass of DNA molecules can be collected to be used to form a DNA profile or fingerprint.
You will be treating the cheek cells with salt, liquid detergent and ethanol in order to isolate the DNA. The
material you will get will be white, slimy. 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!
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Materials:
 Plastic test tube                                             plastic wrap
 3 ml plastic pipettes with suction bulb                       small, plastic pipette
 8% sodium chloride solution                                   Alcohol (95% ethyl or isopropyl)-chilled
 beaker to hold test tube                                      Toothpick or plastic mini-pipette
 Gatorade, graduated cylinders                                 1-3 dram, screw-top vial
 bathroom drinking cup
 10 % sodium lauryl sulfate solution (dish
   detergent)
Directions: 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 clean pipette, add 2 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.
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 (dish detergent) 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!
PRECIPITATE THE DNA:
       8) 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.
       9) 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 clumps of white you are observing are
masses
            of DNA molecules!
COLLECT THE DNA:
       10) Carefully use a clean toothpick or the mini-pipette 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!

QUESTIONS: Use information from your notes and the first page of this lab to answer the questions.
           Do not copy the information directly from the packet, 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 molecule, 46 molecules,
   thousands of molecules, etc.) Explain your answer.
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3) What was the role of the salt in this experiment? Explain how it works.
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4) What was the role of the detergent in this experiment?
_________________________________________________________________________________________
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5) What was the purpose of pouring the cold alcohol over the cheek cell solution?
__________________________________________________________________________________________
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6) Would the DNA appear different in the alcohol vial if we had used plant cells instead of human cells?
Explain by describing the difference between plant and animal DNA. Are the differences visible?
__________________________________________________________________________________________
__________________________________________________________________________________________
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____________________________________________________
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posted:2/25/2012
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