Your profession is your passion. Pass it on. - PowerPoint PPT Presentation

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Your profession is your passion. Pass it on.

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Your profession is your passion. Pass it on.
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Your profession is your passion. Pass it on.

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  1. Your profession is your passion. Pass it on.

  2. Genes in a Bottle Activity Today’s Activity • Focuses on isolating your own DNA from cheek cells • The DNA will be transferred into an amulet • The amulet will be a permanent keepsake of your genetic material — Your personal essence!

  3. Genes in a Bottle Activity Timeline Introduction Background on DNA Extraction Extract Genomic DNA from Cheek Cells Prepare DNA Necklaces

  4. Relevance of DNA Isolation Isolation of DNA is often the first step before further analysis • DNA profiling • Cloning • Disease diagnosis • DNA sequencing • Creation of genetically modified organisms (GMO) — agriculture, pharmaceuticals • Environmental testing, biodefense

  5. Cell Bio 101 What are the parts of an animal cell?

  6. Protocol Highlights Step 1: Use a simple water mouth wash to collect cheek cells Step 2: Add lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents Step 3: Precipitate DNA with cold alcohol in high salt Step 4: Transfer your DNA into the amulet

  7. Illustrated Guide of Kit Components Plastic transfer pipet 15ml conical tube Micro test tube Amulet

  8. Workstation Inventory Zip-Top Bag Components • 15 ml conical tube 1 • Colored micro test tube with protease/salt/lysis buffer 1 • Disposable plastic transfer pipet 1 • Necklace components 1 • Tattoo 1 • Small paper cup 1

  9. Step 1: Cheek Cell Collection In this step, you will collect your cheek cells in a 15 ml conical tube using a water mouth wash. Take out the 15 ml conical tube which contains 3 ml of water and label with your initials. Practice using the transfer pipet by transferring 1 ml of water from a large cup to your small cup. Repeat 3 times for a total of 3 ml of water. Take the water from the small cup into your mouth — don’t swallow it! Swish the water around like mouthwash and gently chew on the inside of your cheeks while you swish the water around for 30–60 seconds. Carefully expel the water back into the small paper cup. Pour the water-cheek cell mix back into your 15 ml tube.

  10. Step 2: Cell Lysis and Proteolysis In this step, you will lyse your cheek cells and degrade the protein with a protease. Open the colored micro test tube containing a protease/salt/lysis buffer mixture. Pour the entire contents into the 15 ml conical tube containing your cheek cells. Place the cap on the tube and gently invert it 5 times — don’t shake it! Observe your tube — do you notice any changes? Incubate for 5 minutes by warming the tube in your hand. This allows time for the protease to break down the proteins.

  11. Step 3: DNA Precipitation In this step, you will precipitate your DNA with the addition of alcohol. Hold your tube at a 45°angle and slowly fill the tube with alcohol by gently pouring or pipetting alcohol down the inside wall of the tube. Fill the tube to the 14 ml mark. Cap your tube and let stand undisturbed for 5 minutes at room temperature. What do you see? (You should begin to see bubbles and white strands appearing at the interface between the alcohol and water phases —this is your DNA!) Very gently tilt the tube on its side and then turn it upright about 10 times until the water and alcohol phases are mixed. Once mixed, the DNA should be fully visible as a “precipitate”.

  12. Step 4: Necklace Assembly In this step, you will assemble your DNA necklace. Place the amulet on your desk. Using a plastic pipet, carefully transfer as much of your DNA precipitate and only as little alcohol as you can into the amulet. The amulet requires approximately 0.5 ml to fill. Screw the cap onto the amulet. Slip the waxed cord through the cap. Your DNA necklace is now complete!

  13. Genes in a Bottle Kit Why Perform Each Step?

  14. Step 1 Cell Collection • How are cells collected? • Gently chewing the inside of the mouth combined with a water mouth wash is used to dislodge epithelial cells lining the mouth. • Ample cell collection is critical for success.

  15. CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 O O O S - O SDS Step 2 Lysis Buffer • What is lysis buffer? • 50 mMTris-HCI, pH 8.0 • 1% SDS • What does each component do? • Tris buffer maintains the pH of the solution at a level where DNA is stable • 1% SDS breaks open the cell and nuclear membranes, allowing the DNA to be released into the solution. SDS also denatures and unfolds proteins, making them more susceptible to protease cleavage Na+

  16. Step 2 Protease/Salt • Why add protease? • Protease is added to destroy nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA • Protease treatment increases the amount of intact DNA that is extracted • Why add salt? • The protease solution already contains salt (NaCl) • Na+ ions of NaCI associate with the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules • The addition of salt allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added

  17. DNA Structure

  18. Step 3 Alcohol • Why add ice cold alcohol? • DNA does not dissolve in alcohol • The addition of cold alcohol makes the DNA clump together and precipitate out of solution • Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands • Microscopic oxygen bubbles “aggregate”, or “fuse” together, as the DNA precipitates • The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the organic phase

  19. Genes in a Bottle™ Kit Congratulations! You have just created your very own DNA necklace!

  20. How long does the DNA in the necklace last? The DNA in the amulet can last for years. Add more alcohol into the vial if some evaporation occurs. Bulletin 6279 Rev B 13-0497 0313