Transcript PowerPoint presentation for younger students - Bio-Rad

Your profession is your passion. Pass it on.
bio-rad.com/scienceambassadors
Bio-Rad Science Ambassador Program
Genes in a Bottle Activity
Today’s Activity
 Focuses on isolating your own DNA from cheek cells
 The DNA will be transferred into an amulet
 The amulet will be a permanent keepsake of your genetic material —
Your personal essence!
2
Bio-Rad Science Ambassador Program
Genes in a Bottle Activity
Timeline
Introduction
Background on DNA Extraction
Extract Genomic DNA from Cheek Cells
Prepare DNA Necklaces
3
Bio-Rad Science Ambassador Program
Relevance of DNA Isolation
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
4
Bio-Rad Science Ambassador Program
Cell Bio 101
What are the parts of an animal cell?
5
Bio-Rad Science Ambassador Program
Protocol Highlights
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 amulet
6
Bio-Rad Science Ambassador Program
Illustrated Guide of Kit Components
Plastic transfer pipet
Micro test tube
7
Bio-Rad Science Ambassador Program
15ml conical tube
Amulet
Workstation Inventory
Zip-Top Bag Components
8
15 ml conical tube
1
Colored micro test tube with protease/salt/lysis buffer
1
Disposable plastic transfer pipet
1
Necklace components
1
Tattoo
1
Small paper cup
1
Bio-Rad Science Ambassador Program
Step 1:
Cheek Cell Collection
In this step, you will collect your cheek cells in a 15 ml conical tube using a water mouth wash.
1. Take out the 15 ml conical tube which contains 3 ml of water and
label with your initials.
2. Practice using the transfer pipet by transferring 1 ml of water from
a large cup to your small cup. Repeat 3 times for a total of 3 ml of
water.
3. Take the water from the small cup into your mouth — don’t
swallow it! Swish the water around like mouthwash and gently
chew on the inside of your cheeks while you swish the water
around for 30–60 seconds.
4. Carefully expel the water back into the small paper cup.
Pour the water-cheek cell mix back into your 15 ml tube.
9
Bio-Rad Science Ambassador Program
Step 2:
Cell Lysis and Proteolysis
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.
10
Bio-Rad Science Ambassador Program
Step 3:
DNA Precipitation
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”.
11
Bio-Rad Science Ambassador Program
Step 4:
Necklace Assembly
In this step, you will assemble your DNA necklace.
1. Place the amulet on your desk. Using a plastic
pipet, carefully transfer as much of your DNA
precipitate and only as little alcohol as you
can into the amulet. The amulet requires
approximately 0.5 ml to fill.
2. Screw the cap onto the amulet.
3. Slip the waxed cord through the cap. Your DNA
necklace is now complete!
12
Bio-Rad Science Ambassador Program
Genes in a Bottle Kit
Why Perform Each Step?
13
Bio-Rad Science Ambassador Program
Step 1
Cell Collection
How are cells collected?
 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.
14
Bio-Rad Science Ambassador Program
Step 2
Lysis Buffer
CH3
CH2
What is lysis buffer?
 50 mM Tris-HCI, pH 8.0
 1% SDS
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
S
-
O
O
O
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
Na+
SDS
15
Bio-Rad Science Ambassador Program
Step 2
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
16
Bio-Rad Science Ambassador Program
DNA Structure
17
Bio-Rad Science Ambassador Program
Step 3
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
18
Bio-Rad Science Ambassador Program
Genes in a Bottle™ Kit
Congratulations!
You have just created your
very own DNA necklace!
19
Bio-Rad Science Ambassador Program
How long does the DNA in the necklace last?
The DNA in the amulet can last for years.
Add more alcohol into the vial if some
evaporation occurs.
Bulletin 6279 Rev B
20
Bio-Rad Science Ambassador Program
13-0497
0313