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Bio-Rad Science Ambassador Program

Today’s Activity
 Focuses on isolating your own DNA from cheek cells
 The DNA will be transferred into a glass amulet
 The amulet will be a permanent keepsake of your genetic material —
Your personal essence!
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Timeline
Introduction
Background on DNA Extraction
Extract Genomic DNA from Cheek Cells
Prepare DNA Necklaces
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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
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What are the parts of an animal cell?
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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 glass amulet
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Zip-Top Bag Components
15 ml tubes each containing 3 ml water
Colored micro test tube with protease/salt/lysis buffer
Disposable plastic transfer pipet
Necklace components
Tattoo
Small paper cup
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1
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In this step, you will collect your cheek cells in a 15 ml conical tube using a water mouth wash.
1.
Label the 15 ml conical tube containing
3 ml of water with your initials
2.
Take the water from the 15 ml conical tube into your mouth — don’t swallow it!
Chew gently on the inside of your cheeks while you swish the water around for
30
–60 seconds.
3.
Carefully expel the liquid back into the small paper cup.
4.
Pour the water-cheek cell mixture back into your 15 ml tube.
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In this step, you will lyse your cheek cells and degrade the protein with a protease.
1.
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.
2.
Place the cap on the tube and gently invert it
5 times — don’t shake it! Observe your tube — do you notice any changes?
3.
Incubate for 5 minutes by warming the tube in your hand. This allows time for the protease to break down the proteins.
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In this step, you will precipitate your DNA with the addition of alcohol.
1.
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.
2.
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!)
3.
Very gently tilt the tube on its side and then turn it upright about 10 times until the water and alcohol phases are mixed.
4.
Once mixed, the DNA should be fully visible as a “precipitate”.
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In this step, you will assemble your DNA necklace.
1.
Using a disposable plastic transfer pipet, carefully transfer as much of your DNA and as little alcohol as possible into your glass vial.
Fill the vial to no more than 2 mm from the top.
2.
Firmly push the plastic stopper cap into the neck of the vial to seal the glass vial.
3.
Have an adult apply a small drop of super glue to the inside of the silver cap and press it onto the vial. Allow to dry for several minutes.
4.
Slip the waxed cord through the silver cap.
Your DNA necklace is now complete!
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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.
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Lysis Buffer
What is Lysis Buffer?
 50 mM Tris-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
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CH
3
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
O
CH
2
O
Na +
O
S
-
SDS
O

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
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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
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Congratulations!
You have just created your very own DNA necklace!
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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.
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Bulletin 6279 Rev A 12-0295 0512