Lab method for extracting DNA from human CHEEK CELLS
See Your DNA = Crack the Code of Life
Materials
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Light colored Gatorade = 10 ml
Liquid detergent = 1 ml
Ethyl Alcohol (COLD) = 5 ml (Put on ICE)
1 plastic cup
1 - 50 ml beakers
1 - Graduated cylinder - small
Glass stirring rod or wooden stick for stirring mixture
Eye dropper or paper towel – may be needed to remove foam
Paper clip hook for collecting DNA
1.
Pour 10 milliliters of Gatorade into a plastic cup. Take the 10 ml Gatorade and vigorously swish it
in your mouth, making sure to rub your tongue along your cheeks and chew on your cheek (use
your teeth, but NO BLOOD) for 30 seconds. This amount of swishing will actually become quite
laborious—hang in there! Just remember: “spit happens”.
2.
CAREFULLY spit Gatorade back into cup; then pour 10 ml of spit into clean 50 ml beaker.
3.
Add 1 milliliter (15 – 18 drops) of liquid detergent to the beaker.
4.
Using stirring rod gently mix the contents for 5 minutes. DO NOT MAKE FOAM. Use paper towel
to remove foam that might be made. (The detergent will break open the cell membrane to release
the DNA into the soap solution. Do not be too vigorous while mixing! DNA is a very long molecule.
Physical abuse can break it into smaller fragments, a process known as shearing.)
5.
Slightly tilt beaker and pour 5 ml fluid ounces of the chilled
ethyl alcohol SLOWLY down the side of the beaker so that
it forms a layer on the top of your soapy solution. DO NOT TIP,
SHAKE, OR MIX THE BEAKER or you may not see the DNA.
Put beaker down on table and then ”get down” and look.
6.
Wait for about 5 minutes and watch as the DNA floats to the surface. If you look to the line of
separation between the two layers, or the interface, you will start to see bubbles attached with tiny
hair like white strings. These strings are your DNA
7.
Use the glass rod or wooden stick to remove DNA from beaker by twirling the rod or stick in a
circular motion. Be careful to minimize mixing of alcohol and soapy layers. If too much shearing
has occurred, the DNA fragments may be too short to wind up, and they may form clumps instead.
Place on paper towel and dry DNA OR
8. If time permits, place small sample of DNA on slide; look at it with microscope.
Be sure to share results with all lab partners!!!
TEACHER SIGNATURE: CLEAN UP!!!!
DNA EXTRACTION
Name________________ Table#_____
Group Names: ______________________
______________________
Date________________ Per._____
______________________
______________________
50% of your Grade
is the DNA Product
Teach
sign
READ NOTES ON BACK
1. List three things available to you that you might use for a DNA source besides wheat germ, onion,
cheek cells or split peas.
2. List three things that would not be a DNA source.
3. What is the role/purpose of DNA in a cell?
4. a. Explain how DNA is contained in a cell.
b. Why does DNA extraction need to be performed in order to obtain DNA?
5. a. Describe what you saw happening in the beaker.
b. Describe the product that was obtained.
6. What is an Enzyme?
7. Why did you use detergent in the procedure, and how does it work? (see notes on back)
8. What possible benefits might be obtained by the ability to isolate the DNA of any organism?
9. Which “ingredient” (besides wheat germ,cheek cells, onions, or peas) do you think might have the most
effect on the amount of DNA you extract? __________________________________________
10. How can you test your Hypothesis? Describe an experiment to test your hypothesis.
HOW DOES IT WORK?
DNA is present in all living things from bacteria to plants to
animals. In animals, it is found in almost all cell types: cheek, muscles, reproductive cells, hair roots, - anything with a nucleus. DNA is NOT found in Red blood cells because they lack nuclei. White
blood cells do have a nucleus. DNA in a cell is about 100,000 times as long as the cell itself.
However, DNA only takes up about 10% of the cell's volume. How can this be? This is because the
DNA molecules fold themselves many times to pack themselves in the cell's nucleus.
Each chromosome contains a single immense molecule of DNA that, in humans, has a length of up to
12 centimeters when stretched out! (look at 12 cm on ruler) As a matter of fact, all the DNA in one
human cell (on all 46 chromosomes) is about two meters long, yet fits into a cell nucleus which is 2-3
micrometers (that's .000002 meters wide!). Yet, the DNA must still be in such a state as to allow for
enzymes to replicate the molecule or initiate the production of a protein. The 23 pairs of human
chromosomes are estimated to include about 100,000 genes.
WHEAT GERM
Wheat germ comes from wheat seeds. The “germ” is
the embryo, which is the part of the seed that can
grow into a new wheat plant. When wheat seeds are
milled into white flour, the wheat germ and wheat bran
are removed, leaving only starch. Wheat germ
contains many nutrients while wheat bran consists of
fiber. Whole-wheat flour contains all parts of the
wheat seed and is therefore more nutritious than white flour while also providing
important fiber for digestion.
CHEEK CELLS
Cheek cells come from the inner lining of mouth or the check.
These cells are routinely shed and replaced by new cells.
As the old cells die, they accumulate in the saliva in the
mouth and can easily be collected by using mouthwash.
One might say “Spit Happens”.
ONION CELLS
An onion is used because it has a low starch content, which allows DNA to be seen.
The walls of onion cells are made of cellulose, which is a polysaccharide made of
glucose. Cellulose provides a tough barrier that protects
the cell. In order to examine the contents of a plant cell,
blending helps to burst open the cell's walls and release
contents. Onion cells also have membranes that envelop
their contents. Cell membranes are made of a double
layerof lipids that allow the movement and transfer of certain
ions and substances in and out of the cell. Breaking down a
cell's membrane allows you examine the contents of the cell.
Detergents contain chemical compounds that can break down cell membranes.
Water temperature
The heat softens the phospholipids (fats) in the membranes that surround the cell and the nucleus.
It also inactivates (denatures) the deoxyribonuclease enzymes (Dnase) which, if present, would cut
the DNA into such small fragments that it would not be visible. Denatured enzymes and DNA
unravel, loose their shape, and thus become inactive. Enzymes denature at 60°C and DNA
denatures at 80°C.
Detergent
Detergent contains sodium laurel sulfate, which cleans dishes by removing fats and proteins. It
acts the same way in the DNA extraction, pulling apart the fats (lipids) and proteins that make up the
membranes surrounding the cell and the nucleus. Once these membranes are broken apart, the
DNA is released from the cell.
Soap molecules and grease molecules are made of two parts:
Hydrophilic heads: which LIKE water and Hydrophobic tails which HATE water
Both soap and grease molecules organize themselves in bubbles (spheres) with heads outside to
face the water and tails inside to hide from the water.
When soap comes close to grease, it captures it,
forming a greasy soapy ball:
A cell’s membrane has two layers of lipid (fat) molecules with proteins between them:
When detergent comes close to cell, it captures the lipids & proteins & releases DNA:
Alcohol
The DNA released from the cell nucleus is dissolved in the water/detergent/wheat germ solution and
cannot be seen. DNA precipitates out of solution in alcohol, where it can be seen. Besides allowing
us to see the DNA, the alcohol separates the DNA from the other cell components, which are left
behind in the water solution. The alcohol also causes gases dissolved in the water to be released,
which may be observed as small bubbles.
Meat Tenderizer : acts as an enzyme to cut proteins just like a
pair of scissors. The DNA in the nucleus of the cell is molded, folded,
and protected by proteins. So, the enzyme (papain) cuts the proteins
away from the DNA. Papain also helps break down DNAase, an enzyme
that breaks down DNA)
(Not used in all of the DNA extractions)
Results Questions:
1. What does the salt do? (gatorade) (Salt provides the DNA with a favorable environment; it contributes
positively charged atoms that neutralize the normal negative charge of DNA.)
2. What does the blender do? (help break down the cell walls)
3. When you mix the blended cell source with the soap, what is happening? (In the experiment, the enzymes
in the soap are breaking down the lipid molecules of the cell and nuclear membranes, releasing the contents of
the cell, including the DNA. These enzymes in the soap are what break down grease while washing dishes.)
4. What does the alcohol do? Why does the DNA rise to the top after adding alcohol?
(DNA will not dissolve in this alcohol, so the DNA comes out of the solution, or precipitates. It is less dense
than water or cell scum--which is what settles to the bottom of the glass--so it floats up into the alcohol layer,
where you see it as a snotty, string-like substance, with small bubbles formed on it.)
5. If you try a seed food such as peas, there will be more protein residue in the liquid. Why? (Because
protein is stored in them for the nutrition of the new plant.)
6. Why can’t you see the double helix? (It is too small to be seen with the naked eye. What you extracted is
millions of strands of DNA.)
7. What part of the cell did the DNA come from? (99% is from the nucleus.)
BONUS:
1. If you did the experiment with both plant and animal cells, how would their DNA compare?