Presentation on theme: "Biotechnology Explorer Program Serious About Science Education."— Presentation transcript:
Biotechnology Explorer Program Serious About Science Education
Genes in a Bottle Kit : Capture Your Unique Essence! EDU Kirk Brown, Tracy High School, Tracy, CAKirk Brown, Tracy High School, Tracy, CA Stan Hitomi,,Stan Hitomi, Edward Teller Education Center, University of California, CA
Genes in a Bottle Workshop Timeline Introduction Background on DNA extraction Extract genomic DNA from cheek cells Prepare DNA necklaces Review extension experiments
Why teach DNA Extraction? Powerful teaching tool Powerful teaching tool Real-world connections Real-world connections Link to careers and industry Link to careers and industry Tangible results Tangible results Laboratory extensions Laboratory extensions
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
Middle Schools (grades 5-8) Secondary/High Schools (grades 9-12) 2-year and 4-year Colleges Genes in a Bottle kit is Suitable for :
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 experimentElectrophorese DNA samples on an agarose gel as an extension experiment
Cell Bio 101 What are the structures of the cell? What are the structures of the cell?
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.
Flow chart Activity
Teacher's (Common) Station Water bath at 50 o C 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 buffer4 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 chloride1 Clear, capless screw cap tubes1 Assorted colored screw caps4 Cytology brushes8 5 ml round-bottom test tubes4 Parafilm (small pre-cut pieces)4 Disposable plastic transfer pipets4 Foam micro test tube holder1 Permanent marker1 Disposable paper cup or beaker for waste collection1 DNA Extraction and Precipitation Workstation set up 4 students/workstation for a total 36 students
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.
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.
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. 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. Gently invert 5 times to mix. 11. Incubate sample in a 50 C water bath for 10 minutes.
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.
Laboratory Protocol 15. Slowly add a pipetful of cold alcohol, holding the tube a 45 angle. 16. Let stand undisturbed for 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.
Why perform each step?
1. Cell collection Two soft-bristled brushes are used to dislodge epithelial cells lining the mouth. Ample cell collection is very critical for success.
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 O S O O O - CH 2 CH 3 SDS
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.
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.
Na + O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar O
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
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
2.Firmly push the plastic stopper cap into the neck of the vial to seal the glass vial 3.Slip the waxed cord through the silver cap 4.Apply a small drop of Super glue to into the inside of the silver cap. 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 minutes and then check for complete seal 6. After the glue has dried, tie the waxed cord. DNA necklace preparation continued…
Congratulations! You have just created your very own DNA Necklace !
The DNA in the glass vial can last for years. Add more alcohol into the vial if some evaporation occurs. How long does the DNA in the necklace last?
Genes in a Bottle ( EDU) Kit Components Kit Provides Enough Materials for 36 Students Contains one DNA Extraction Module ( EDU) two DNA Necklace Modules ( EDU) DNA Extraction Module ( EDU): (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 DNA Necklace Module ( EDU): (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 Recommended (Optional) Accessories: Water bath with thermometer EDU Required Accessories Not Included in Kit: 91% isopropanol (available from drugstores) or 95% ethanol, 250 ml Container of ice, 1 Permanent marker, 1–8