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  1. Biotechnology Explorer Program Serious About Science Education

  2. Genes in a Bottle Kit : Capture Your Unique Essence! 166-2300EDU • Kirk Brown, Tracy High School, Tracy, CA • Stan Hitomi, Edward Teller Education Center, • University of California, CA

  3. Genes in a Bottle Workshop Timeline • Introduction • Background on DNA extraction • Extract genomic DNA from cheek cells • Prepare DNA necklaces • Review extension experiments

  4. Why teach DNA Extraction? • Powerful teaching tool • Real-world connections • Link to careers and industry • Tangible results • Laboratory extensions

  5. Why use Bio-Rad’s Genes in a Bottle Kit? • Provides enough reagents for 36 student DNA extractions • The activity fits into a single 50-minute period • Curriculum Manual is set up into two sections: Basic level instruction (for 5-8 grade) Advanced level instruction (for 9-14 grade) • This activity does not require any specialized equipment • The product is accompanied by Bio-Rad’s world-class technical support

  6. Genes in a Bottle kit is Suitable for : • Middle Schools (grades 5-8) • Secondary/High Schools (grades 9-12) • 2-year and 4-year Colleges

  7. What can you do with the Genes in a Bottle Kit ? • Reinforce the structures of a cell • DNA structure and function • Enzyme function • Introduce students to procedures involved in DNA extraction • Preserve student DNA samples in DNA necklaces • Electrophorese DNA samples on an agarose gel as an extension experiment

  8. Cell Bio 101 • What are the structures of the cell?

  9. Protocol Highlights: Genomic DNA Extraction • Use 2 soft bristled brushes to collect cells from the inside of each cheek, and the space between lips and gums. • Add cells to lysis buffer to break open cell and nuclear membranes and to release nuclear contents. • Digest sample with protease to degrade proteins. • Precipitate DNA with cold alcohol in high salt environment.

  10. Activity Flow chart

  11. DNA Extraction and Precipitation Workstation set up 4 students/workstation for a total 36 students Teacher's (Common) Station Water bath at 50oC Ice-cold bottle of 91% isopropanol or 95% ethanol on ice Students’ Workstation (4 students per station) Number required Clear micro test tubes, each containing 1 ml lysis buffer 4 Blue micro test tube labeled “prot”, containing 250 ul of pre-diluted protease 1 Pink micro test tube labeled “salt”, containing 250 ul of sodium chloride 1 Clear, capless screw cap tubes 1 Assorted colored screw caps 4 Cytology brushes 8 5 ml round-bottom test tubes 4 Parafilm (small pre-cut pieces) 4 Disposable plastic transfer pipets 4 Foam micro test tube holder 1 Permanent marker 1 Disposable paper cup or beaker for waste collection 1

  12. Ample cell collection is very critical for success. For best results, make sure to spend the recommended amount of time collecting and carefully transferring the cells into the tube of lysis buffer.

  13. Laboratory Protocol 1. Label one clear microtube containing Lysis buffer (labeled “lysis”) with your initials. 2. Roll the first brush inside your left cheek for 1 minute and continue scrapping between your cheek and gum and the roof of the mouth. 3. Place the brush into tube of Lysis buffer and rotate the brush vigorously to release cells. 4. Scrape on top of tube to get remaining cells off. Put the brush in a waste container.

  14. Laboratory Protocol 5.Obtain the second brush. Roll the brush inside your right cheek for 1 minute and continue scrapping between your cheek and gum and the roof of the mouth. 6. Place the brush into your tube of Lysis buffer and rotate the brush vigorously to release cells. 7. Scrape on top of tube to get remaining cells off. Put the brush in a waste container.

  15. Laboratory Protocol 8. Cap microtube containing cheek cells and buffer, and gently invert 5 times to mix. 9. Add 1 drop of Protease (labeled “prot”) to your sample. 10. Gentlyinvert 5 times to mix. 11. Incubate sample in a 50C water bath for 10 minutes.

  16. Laboratory Protocol 12. Using a clean plastic transfer pipet, add 2 drops of salt to your microtube . 13. Gently invert microtube 5 times to mix 14. Transfer (pour) your cell extract into a 5 ml tube.

  17. Laboratory Protocol 15. Slowlyadd a pipetfulof cold alcohol, holding the tube a 45 angle. 16. Let stand undisturbedfor 5 minutes at room temperature. What do you see? 17. Cover 5 ml tube with parafilm, and gently invert 10 times to bring DNA out of solution.

  18. Why perform each step?

  19. 1. Cell collection Two soft-bristled brushes are used to dislodge epithelial cells lining the mouth. Ample cell collection is very critical for success.

  20. 2. Lysis buffer • What is in the Lysis Buffer? • 50 mM Tris-HCl, pH 8.0 • 1% SDS • Tris buffer to maintain the pH of the solution at a level where DNA is stable • 1% SDS to break open the cell and nuclear membranes, allowing the DNA to be released into the solution • SDS also functions to denature and unfold proteins, making them more susceptible to protease cleavage CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 O S O O - O SDS

  21. 3.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.

  22. 4. Adding salt (5M NaCl) • Na+ ions of NaCl bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules. • The addition of NaCl 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.

  23. O Na+ O P O Base Na+ O O CH2 Sugar O Na+ O O P Base Na+ O O CH2 Sugar OH

  24. 5. Adding ice cold alcohol? • DNA cannot 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, simultaneously with the DNA precipitation • The larger, visible air bubbles act to “lift” the DNA out of solution, from the aqueous into the organic phase

  25. Preserving DNA sample: DNA necklace preparation 1. Using a plastic transfer pipet, carefully transfer the fluffy DNA strands you extracted into the small glass vial -- Transfer as much of your DNA and as little alcohol as possible -- The vial should be filled no higher than ½ cm from the top of the neck of the vial

  26. DNA necklace preparation continued… • Firmly push the plastic stopper cap into the neck of the vial to seal the glass vial • Slip the waxed cord through the silver cap • Apply a small drop of Super glue to into the inside of the silver cap.

  27. DNA necklace preparation continued… 5. Place the silver cap onto the top of the glass vial and press down firmly for 30 seconds. Allow the glue to dry for 10-15 minutes and then check for complete seal 6. After the glue has dried, tie the waxed cord.

  28. Congratulations! You have just created your very own DNA Necklace !

  29. How long does the DNA in the necklace last? The DNA in the glass vial can last for years. Add more alcohol into the vial if some evaporation occurs.

  30. Genes in a Bottle (166-2300EDU) Kit Components • Kit Provides Enough Materials for 36 Students • Contains oneDNA Extraction Module (166-2000EDU) twoDNA Necklace Modules (166-2200EDU) DNA Necklace Module (166-2200EDU): (contains enough material for 18 DNA necklaces; order 2 modules for a classroom of 36 students) Glass vials, 18 Silver caps, 18 Plastic plugs, 18 Waxed string, 18 Super glue gel, 1 tube Instruction manual DNA Extraction Module (166-2000EDU): (contains enough material for 36 students) Lysis buffer, 40 ml Protease, 1.3 ml 5 M sodium chloride (salt), 5 ml Sterile water, 2.5 ml 5 ml round bottom tubes with no lid, 50 Clear, flip-top microtubes, 30 Multicolor, flip-top microtubes, 60 Clear, capless screwcap tubes with assorted color caps, 40 Disposable plastic transfer pipets, 50 Foam microtube holders, 10 Cytology brushes, 80 Parafilm, 1 strip Curriculum, including a teacher’s guide, and a graphic quick guide, and separate student instructions for basic and advanced-level instruction Required Accessories Not Included in Kit: 91% isopropanol (available from drugstores) or 95% ethanol, 250 ml Container of ice, 1 Permanent marker, 1–8 Recommended (Optional) Accessories: Water bath with thermometer 166-0504EDU