2. Capture Your Unique Essence!
Genes in a Bottle
Capture Your Unique Essence!

3. Genes in a Bottle Kit Capture Your Unique Essence! Instructors
Stan Hitomi
Coordinator – Math & Science
San Ramon Valley Unified School District
Danville, CA
Kirk Brown
Lead Instructor, Edward Teller Education Center
Science Chair, Tracy High School
and Delta College, Tracy, CA
Sherri Andrews, Ph.D.
Curriculum and Training Specialist
Bio-Rad Laboratories
Essy Levy, M.Sc.

4. Why Teach DNA Extraction?
• Links classroom to real-world science
• Tangible results
• Laboratory extensions
- cheek cell staining with Fast Blast DNA Stain
(an excellent microscope activity)
- staining precipitated DNA with Fast Blast DNA Stain
- DNA electrophoresis/fingerprinting
Aligned with standards
- Reinforce the structures of the cell
- DNA structure and function
- Enzyme function

6. Genes in a Bottle Kit Advantages
• Provides enough reagents for 36 student DNA extractions
• The activity fits into a single 45-minute period
• Curriculum Manual is set up in two sections:
• Basic level instruction (for grades 5-8)
• Advanced level instruction (for grades 9-14)
• This activity does not require any specialized equipment
• The product is accompanied by Bio-Rad’s world-class technical support
Cost effective
Raise $$ for science programs

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

8. Relevance of DNA Isolation
Isolation of DNA is often the first
step before further analysis
DNA profiling
Cloning
Disease diagnosis
DNA sequencing
Genetically modified organisms (GMO) -agriculture, pharmaceutical
Environmental testing, biodefense
DNA extraction is one of the first steps to many research, forensic or clinical analyses. Extracting one's own DNA and making it visible by precipitation is a visible and tangible way to explore DNA.

9. Cell Bio 101: What are the Structures of the Cell?
This experiment fits well into exploring cell structures: Where is the DNA located? What properties of cell membranes allow us to break them open with detergents? Why is protease used to assist in extraction of DNA? Further discussion later in workshop.

10. Cell Structures
Lysozome

11. Cell Structures
Chloroplasts in a plant cell. The dark, oval shapes are starch granules within the chloroplasts.

12. Protocol Highlights: Genomic DNA Extraction
• Use a simple water mouthwash to collect cheek cells
• Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
• Digest sample with protease to degrade proteins
• Precipitate DNA with cold alcohol in high salt
Overview of main steps in experiment.

13. Genes in a Bottle: Procedure Overview
Graphic overview chart

14. DNA Extraction and Precipitation Workstation Inventory 4 Students/Workstation for a total of 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) No. Required
15 ml tubes each containing 3 ml water 4
Pink micro test tube labeled “prot”,
containing 1.25 ml of rehydrated protease + salt 1
15 ml tube labeled ‘lysis’ containing 10 ml Lysis buffer 1
Disposable plastic transfer pipets 6
Foam micro test tube holder 1
Permanent marker 1
Disposable paper cup or beaker for holding 15 ml tubes
and subsequent waste collection 1
Necklace components or 1.5 ml flip-top
tubes for DNA storage 1
Quick Guide 1
For efficient time management, begin the extraction protocol and discuss experiment during incubation times. The student workstations have the indicated items for a group of 4 students per workstation.
The protease must be rehydrated before use; once rehydrated, the protease will remain active for several weeks if stored at 4oC.

15. Ample Cell Collection is Critical for Success
For best results, make sure students spend the recommended amount of time collecting mouth cells.
Some users may find collecting mouthwash in a 15 ml tube difficult.
As an alternative, instructors may wish to use a small drinking cup to dispense and collect mouth wash.
This step cannot be stressed enough. Best results happen when class collects cells as a group, with one person keeping time. The instructor can "cheerlead" to encourage ample cells are collected. For teachers that have done DNA extraction from strawberry or thymus, fewer cells collected in this experiment. It is imperative that sufficient cells are collected.

16. Laboratory Quick Guide

17. Laboratory Protocol
Obtain 15 ml tube containing 3 ml water from your instructor. Label the tube with your initials.
2. Gently chew the insides of your cheeks for 30 seconds. It is NOT helpful to draw blood!
3. Take the water from the 15 ml tube into your mouth, and swish the water around vigorously for 30 seconds.
4. Carefully expel the liquid back into the 15 ml tube.
After everyone labels a 15 ml tube containing water with their initials (use permanent marker and label the tube on its side), begin collecting as a group.
The hardest part of this exercise will be to expel the mouth wash back into the 15ml tube. You may wish to collect the mouth wash into a cup and then pour the mouth wash into a 15 ml tube instead.

18. Laboratory Protocol
5. Obtain the tube of lysis buffer from your workstation, and add 2 ml of lysis buffer to your tube.
6. Place the cap on the tube, and gently invert your tube 5 times (don’t shake it!). Observe your tube — do you notice any changes? If you do, write them down.
If anyone ends up with a lot more than 5 mls (7 mls or more), they need to remove the excess and discard it. DNA precipitation may not occur optimally if much more than 5 mls of cell lysates are present when alcohol is added during the precipitation step.
It’s important NOT to shake the lysed cells – this will shear the DNA (break it into small fragments) and shorn DNA will not precipitate well.

19. Laboratory Protocol 7. Obtain the tube of protease (prot) at
your workstation. Add 5 drops of
protease to your tube.
8. Place the cap on your tube,
and gently invert it a few
times.
9. Place your tube in a test tube rack or beaker in the water bath and incubate at 50°C for 10 minutes.
It’s important NOT to let students shake the tubes – the DNA will shear, and DNA will not precipitate well if it’s shorn.

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

21. Cell Collection 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.
Epithelial cells are constantly being sloughed off an provide an easily accessible cell supply.
Note: eating food, chewing gum, or dental work just prior to the exercise may affect ample cell collection.
To achieve maximum success (largest DNA precipitates), it’s recommended that people don’t engage in these activities just prior to performing this exercise.

22. 2. Lysis Buffer What is Lysis Buffer. • 50 mM Tris-HCI, pH 8
2. Lysis Buffer What is Lysis Buffer? • 50 mM Tris-HCI, 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 denatures and unfolds proteins, making them more susceptible to protease cleavage). O S -
CH2
CH3
SDS
Can talk about lipid bilayer model of cell membranes and how detergents work.
Na+

23. 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.
Histones, which cause DNA to supercoil (compact) in the chromosome, are proteins.

24. 4. Adding Salt • The protease solution already contains salt
• Na+ ions of NaCI bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules.
• The addition of NaCI 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.

25. DNA Structure O Na+ P Base CH2 Sugar OH
Graphic showing Na+ ions associating with negatively charged phosphate groups on DNA backbone.

26. 5. Adding 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.

27. Prepare stoppers for necklaces
Remove stopper from vial
Trim “ears” off stopper on 2 sides
Replace in vial for later necklace creation
WHY? So the stopper doesn’t create an air pocket preventing the cap from seating fully on the vial

28. Laboratory Protocol
15. Slowly add 10 ml of cold alcohol, holding the tube at a 45° angle. This will take repeated additions using the disposable transfer pipet.
16. Let stand undisturbed for 5 minutes at room temperature. What do you see?
17. Cap your tube, and very gently tilt tube on its side then turn upright about 10 times until both the water and alcohol phases have mixed and the DNA comes out of solution.
It is important to add the alcohol gently and keep tube at a 45 degree angle. After a few minutes, they should see small bubbles with DNA strands attached. If they see a nice clump of DNA, they can remove it to necklace vial before inversion. Alternatively, they can gently mix/tilt their test tube to see the DNA come out of solution and then make a necklace. It's important NOT to shake and dissociate the DNA that is precipitating.

29. Preserving DNA Sample: DNA Necklace Preparation
Using a disposable 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 2 mm from the top of the neck of the vial.
First, remove the plastic stopper from the vial (you will replace the stopper after transferring your DNA). Don’t lose the stopper! It’s small and hard to see.
Next, using a disposable transfer pipet, carefully transfer the DNA strands to the glass amulet. Don’t overfill the amulet; the stopper will be difficult to seal on the vial if there is too much liquid inside.

30. Preserving DNA Sample: DNA Necklace Preparation
Firmly push the trimmed 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 the inside of the silver cap.
Necklace preparation is a little "fiddly" and may be done the next day if class time is limited. Place student samples in refrigerator until next class period if time is limiting.

31. Preserving DNA Sample: DNA Necklace Preparation
Place the silver cap onto the top of the glass vial and press down firmly for 30 seconds. Allow the glue to dry for an additional 10–15 seconds, and then check for a complete seal.
After the glue has dried, tie the waxed cord.
People have a tendency to keep pushing and then pulling on the cap to see if it has dried. I encourage folks to leave their necklaces alone for several minutes without continuously pushing down. It will dry…

32. Congratulations! Genes in a Bottle Kit You have just created your
very own DNA Necklace!

33. How Long Does the DNA in the Necklace Last?
glass vial can last
for years. Add more alcohol into the vial if some evaporation occurs.

34. Genes in a Bottle Kit Components
DNA Extraction
Module ( EDU):
(contains enough material for 36 students)
Lysis buffer, 150 ml
Powdered protease + salt, 1.5 g
15 ml tubes, 50
Clear, flip-top microtubes, 60
Multicolor, flip-top microtubes, 60
Disposable plastic transfer pipets, 60
Foam microtube holders, 10
Curriculum, including a teacher’s guide, 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
( EDU) Kit provides enough materials for 36 Students Above Kit Contains: (1) DNA Extraction Module ( EDU) (2) DNA Necklace Modules ( EDU)
Required Accessories Not Included in Kit:
91% isopropanol (available from drugstores) or 95% ethanol, 360 ml
Container of ice, 1
Permanent marker, 1–8
Recommended (Optional) Accessories:
Water bath with thermometer
EDU