biotechnology explorer program serious about science education
TRANSCRIPT
BiotechnologyBiotechnologyExplorer Program
Serious About Science Education
Genes in a Bottle KitGenes in a Bottle Kit : : Capture Your Unique Essence!Capture Your Unique Essence!
166-2300EDU166-2300EDU
• Kirk BrownKirk Brown, Tracy High School, Tracy, CA, Tracy High School, Tracy, CA
• Stan HitomiStan Hitomi, , Edward Teller Education Center, , University of California, CA
Genes in a Bottle Genes in a Bottle Workshop TimelineWorkshop Timeline
Introduction Introduction
Background on DNA extractionBackground on DNA extraction
Extract genomic DNA from cheek cellsExtract genomic DNA from cheek cells
Prepare DNA necklacesPrepare DNA necklaces
Review extension experimentsReview extension experiments
Why teach DNA Why teach DNA Extraction?Extraction?
Powerful teaching toolPowerful teaching tool Real-world connectionsReal-world connections Link to careers and industryLink to careers and industry Tangible resultsTangible results Laboratory extensionsLaboratory 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 What can you do with the Genes in a Bottle KitGenes 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 Electrophorese DNA samples on an agarose gel as an extension experimentas an extension experiment
Cell Bio 101Cell Bio 101 What are the structures of the What are the structures of the
cell?cell?
Protocol Highlights: Protocol Highlights: Genomic DNA Genomic DNA ExtractionExtraction Use 2 soft bristled brushes to Use 2 soft bristled brushes to collect cellscollect cells
from the inside of each cheek, and the from the inside of each cheek, and the space between lips and gums.space between lips and gums.
Add cells to Add cells to lysis bufferlysis buffer to break open cell to break open cell and nuclear membranes and to release and nuclear membranes and to release nuclear contents. nuclear contents.
Digest sample with Digest sample with proteaseprotease to degrade to degrade proteins.proteins.
Precipitate Precipitate DNA with DNA with cold alcoholcold alcohol in in high high saltsalt environment. environment.
Flow chartActivity
Teacher's (Common) StationWater bath at 50oCIce-cold bottle of 91% isopropanol or 95% ethanol on ice
Students’ Workstation (4 students per station) Number requiredClear micro test tubes, each containing 1 ml lysis buffer4Blue micro test tube labeled “prot”, containing 250 ul of pre-diluted protease 1Pink micro test tube labeled “salt”, containing 250 ul of sodium chloride
1Clear, capless screw cap tubes1Assorted colored screw caps4Cytology brushes 85 ml round-bottom test tubes4Parafilm (small pre-cut pieces)4 Disposable plastic transfer pipets 4Foam micro test tube holder1Permanent marker1Disposable paper cup or beaker for waste collection1
DNA Extraction and Precipitation Workstation set up4 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 ProtocolLaboratory Protocol
1. Label one clear microtube containing Lysis buffer 1. Label one clear microtube containing Lysis buffer (labeled “lysis”) with your initials.(labeled “lysis”) with your initials.
2. Roll the first brush inside your left cheek for 1 2. Roll the first brush inside your left cheek for 1 minute and continue scrapping between your cheek minute and continue scrapping between your cheek and gum and the roof of the mouth.and gum and the roof of the mouth.
3. Place the brush into tube of 3. Place the brush into tube of Lysis bufferLysis buffer and and rotate the brush vigorously to release cells. rotate the brush vigorously to release cells.
4. Scrape on top of tube to get remaining cells off. Put 4. Scrape on top of tube to get remaining cells off. Put the brush in a waste container.the brush in a waste container.
5.5. Obtain the Obtain the secondsecond brush. Roll the brush inside your brush. Roll the brush inside your right cheek for 1 minute and continue scrapping right cheek for 1 minute and continue scrapping between your cheek and gum and the roof of the between your cheek and gum and the roof of the mouth.mouth.
6. Place the brush into your tube of Lysis buffer and 6. Place the brush into your tube of Lysis buffer and rotate the brush vigorously to release cells. rotate the brush vigorously to release cells.
7. Scrape on top of tube to get remaining cells off. Put 7. Scrape on top of tube to get remaining cells off. Put the brush in a waste container.the brush in a waste container.
Laboratory ProtocolLaboratory Protocol
Laboratory ProtocolLaboratory Protocol
8. Cap microtube containing cheek cells and 8. Cap microtube containing cheek cells and buffer, and buffer, and gentlygently invert 5 times to mix. invert 5 times to mix.
9. Add 9. Add 11 drop of Protease (labeled “prot”) to drop of Protease (labeled “prot”) to your sample.your sample.
10. 10. GentlyGently invert 5 times to mix.invert 5 times to mix.
11. Incubate sample in a 5011. Incubate sample in a 50C water bath for 10 C water bath for 10 minutes.minutes.
Laboratory ProtocolLaboratory Protocol
12. Using a clean plastic transfer pipet, add 12. Using a clean plastic transfer pipet, add 2 2 dropsdrops of salt to your microtube . of salt to your microtube .
13. 13. GentlyGently invert microtube 5 times to mix invert microtube 5 times to mix
14. Transfer (pour) your cell extract into a 5 ml 14. Transfer (pour) your cell extract into a 5 ml tube.tube.
Laboratory ProtocolLaboratory Protocol
15. 15. SlowlySlowly add a add a pipetfulpipetful of cold alcohol, of cold alcohol, holding the tube a 45holding the tube a 45 angle. angle.
16. Let stand 16. Let stand undisturbedundisturbed for 5 minutes at for 5 minutes at room temperature. room temperature. What do you see?What do you see?
17. Cover 5 ml tube with parafilm, and 17. Cover 5 ml tube with parafilm, and gentlygently invert 10 times to bring DNA out of solution.invert 10 times to bring DNA out of solution.
Why perform each step?
1. Cell collectionTwo 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
-
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
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+
Na+
Na+
Na+
OCH2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
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 1. Using a plastic transfer pipet, carefully transfer the fluffy DNA strands you transfer the fluffy DNA strands you extracted into the small glass vialextracted into the small glass vial
-- Transfer as much of your DNA and as -- Transfer as much of your DNA and as little alcohol as possiblelittle alcohol as possible
-- The vial should be filled no higher than ½ -- The vial should be filled no higher than ½ cm from the top of the neck of the vialcm 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 10-15 minutes and then check for complete seal
6. After the glue has dried, tie the waxed cord.
DNA necklace preparation continued…
Congratulations! Congratulations! You have just created your very own You have just created your very own
DNA NecklaceDNA 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 (166-2300EDU) Kit Components
Kit Provides Enough Materials for 36 Students Contains one DNA Extraction Module (166-2000EDU)
two DNA Necklace Modules (166-2200EDU) DNA Extraction Module (166-2000EDU):(contains enough material for 36 students)Lysis buffer, 40 mlProtease, 1.3 ml5 M sodium chloride (salt), 5 mlSterile water, 2.5 ml5 ml round bottom tubes with no lid, 50Clear, flip-top microtubes, 30Multicolor, flip-top microtubes, 60Clear, capless screwcap tubes with assorted color caps, 40Disposable plastic transfer pipets, 50Foam microtube holders, 10Cytology brushes, 80Parafilm, 1 stripCurriculum, including a teacher’s guide, and a graphic quick guide, and separate student instructions for basic and advanced-level instruction
DNA Necklace Module (166-2200EDU):(contains enough material for 18 DNA necklaces; order 2 modules for a classroom of 36 students)Glass vials, 18Silver caps, 18Plastic plugs, 18Waxed string, 18Super glue gel, 1 tubeInstruction manual
Recommended (Optional) Accessories:Water bath with thermometer 166-0504EDU
Required Accessories Not Included in Kit:
91% isopropanol (available from drugstores) or 95% ethanol, 250 ml
Container of ice, 1
Permanent marker, 1–8