How to Extract DNA from Organic Matter
There are 4 basic steps to this lab.
1. Physically break down the organic specimen to separate the cells
2. Break down the membranes (lysing) so DNA can get out
3. Cut the DNA away from the proteins
4. Separate the DNA from the rest of the organic matter
You will be adding these things to something with DNA. While there are many things (anything
living) that may be used. We will use __________________.
Step 1: Break apart the cells of the organic matter to make it easier for the chemicals reach them.
This can be done manually with a fork, but we’ll break it down with a blender.
Put these ingredients into a blender:
1. Your DNA source (about 100ml or 1/2 cup of organic material)
2. A large pinch of table salt (less than 1ml or 1/8 teaspoon)
3. Twice as much cold water as the DNA source (about 200ml or 1 cup)
Blend on high for 15 seconds.
Step 2: Break down cell, nuclear, and organelle membranes with detergent (lysing)
Lysing is accomplished with salt (in step 1) and a detergent
Pour the thin ‘soup’ through a strainer (e.g. coffee filter) into a beaker.
Add 30 ml liquid detergent and swirl around to mix.
Let the mixture sit for 8 minutes.
Pour the mixture into test tubes or other small glass containers, each about 1/3 full.
Step 3: Cut the DNA away from the proteins that have been surrounding and protecting the DNA
Add a pinch of enzymes to each test tube and stir gently (so as not to break up the DNA).
In DNA extraction for gel electrophoresis, protein denaturation is accomplished with heat and/or
other chemicals.
In our lab, the chemical papain in meat tenderizer is what separates the DNA from the protein
Step 4: Slowly add alcohol to provide a lighter upper layer for the DNA to move into.
Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the
tube down the side so that it forms a layer on top of the organic mixture. Pour until you have about the
same amount of alcohol in the tube as organic mixture. DNA is soluble in water. But in alcohol it
uncoils and precipitates leaving behind the other cell components below (that are not soluble in
ethanol).
Proteins and fats stay in the bottom organic layer, while DNA floats to the top into the alcohol layer.
You may see them being lifted into the alcohol by bubbles.
You can use a wooden stick or other hook to draw the DNA into the alcohol.
What you have extracted is nucleic acid (DNA and RNA). We cannot use this in gel electrophoresis to
get a DNA fingerprint, because we have extracted all of the DNA (the entire genome). To perform a
gel electrophoresis, you would use restriction enzymes to cut small segments of DNA to be used in
obtaining a DNA fingerprint.
Frequently Asked Questions
1. I'm pretty sure I'm not seeing DNA. What did I do wrong?
First, check one more time for DNA. Look very closely at the alcohol layer for tiny bubbles.
Often, clumps of DNA are loosely attached to the bubbles.
If you are sure you don't see DNA, then the next step is to make sure that you started with
enough DNA in the first place. Many food sources of DNA, such as grapes, also contain a lot of water.
If the blended cell soup is too watery, there won't be enough DNA to see. To fix this, go back to the
first step and add less water. The cell soup should be opaque, meaning that you can't see through it.
Another possible reason for not seeing any DNA is not allowing enough time for each step to
complete. Make sure to stir in the detergent for at least five minutes. If the cell and nuclear membranes
are still intact, the DNA will be stuck in the bottom layer. Often, if you let the test tube of pea mixture
and alcohol sit for 30-60 minutes, DNA will precipitate into the alcohol layer.
2. Why does the DNA clump together?
Single molecules of DNA are long and stringy. Each cell of your body contains six feet of
DNA, but it's only one-millionth of an inch wide. To fit all of this DNA into your cells, it needs to be
packed efficiently. To solve this problem, DNA twists tightly and clumps together inside cells. Even
when you extract DNA from cells, it still clumps together, though not as much as it would inside the
cell.
Imagine this: the human body contains about 100 trillion cells, each of which contains six feet
of DNA. If you do the math, you'll find that our bodies contain more than a billion miles of DNA!
3. Can I use this DNA as a sample for gel electrophoresis?
Yes, but all you will see is a smear. The DNA you have extracted is genomic, meaning that you
have the entire collection of DNA from each cell. Unless you cut the DNA with restriction enzymes, it
is too long and stringy to move through the pores of the gel; instead, all you will end up seeing is a
smear.
4. Isn't the white, stringy stuff actually a mix of DNA and RNA?
That's exactly right! The procedure for DNA extraction is really a procedure for nucleic acid
extraction. However, much of the RNA is cut by ribonucleases (enzymes that cut RNA) that are
released when the cells are broken open.
Other good sources: spinach, chicken liver, onions, broccoli
Put in a blender:
Your DNA source (about 100ml or 1/2 cup of split peas)
A large pinch of table salt (less than 1ml or 1/8 teaspoon)
Twice as much cold water as the DNA source (about 200ml or 1 cup)
Blend on high for 15 seconds.
Why am I adding detergent?
Blending separated the pea cells.
But each cell is surrounded by a sack (the cell membrane). DNA is found inside a secondsack
(the nucleus) within each cell.
To see the DNA, we have to break open these two sacks.
We do this with detergent.
Why detergent? How does detergent work?
Think about why you use soap to wash dishes or your hands. To remove grease and dirt, right?
Soap molecules and grease molecules are made of two parts:
Heads, which like water
Tails, which hate water.
Both soap and grease molecules organize themselves in bubbles (spheres) with their heads
outside to face the water and their tails inside to hide from the water.
When soap comes close to grease, their similar structures cause them to combine, forming a
greasy soapy ball.
A cell's membranes have two layers of lipid (fat) molecules with proteins going through them.
When detergent comes close to the cell, it captures the lipids and proteins.
After adding the detergent, what do you have in your pea soup?
Use meat tenderizer for enzymes. If you can't find tenderizer, try using pineapple juice or contact lens
cleaning solution.
What is an enzyme?
Enzymes are proteins that help chemical reactions happen more quickly. Without enzymes, our
bodies would grind to a halt.
In this experiment, the enzyme we are using comes from meat tenderizer and cuts proteins just
like a pair of scissors.
After the detergent step, the last question was: what do you have now in your pea soup?
The cell and nuclear membranes have been broken apart, as well as all of the organelle
membranes, such as those around the mitochondria and chloroplasts.
So what is left?
Proteins
Carbohydrates (sugars)
DNA
The DNA in the nucleus of the cell is molded, folded, and protected by proteins. The meat
tenderizer cuts the proteins away from the DNA.
The alcohol can be 70-95% isopropyl or ethyl alcohol.
How to Extract DNA from Organic Matter
There are 4 basic steps to this lab.
1. Physically break down the organic specimen to separate the cells
2. Break down the membranes (lysing) so DNA can get out
3. Cut the DNA away from the proteins
4. Separate the DNA from the rest of the organic matter
You will be adding these things to something with DNA. While there are many things (anything
living) that may be used. We will use green split peas.
Step 1: Break apart the cells of the organic matter to make it easier for the chemicals reach them.
This can be done manually with a fork, but we’ll break it down with a blender.
Put these ingredients into a blender:
1. Your DNA source (about 100ml or 1/2 cup of split peas)
2. A large pinch of table salt (less than 1ml or 1/8 teaspoon)
3. Twice as much cold water as the DNA source (about 200ml or 1 cup)
Blend on high for 15 seconds.
Step 2: Break down cell, nuclear, and organelle membranes with detergent (lysing)
Lysing is accomplished with salt (in step 1) and a detergent
Pour the thin pea-cell soup through a strainer (e.g. coffee filter) into a beaker.
How much pea soup do you have?
Add about 1/6 of that amount of liquid detergent (30ml or 2 tablespoons) and swirl to mix.
Let the mixture sit for 5-10 minutes.
Pour the mixture into test tubes or other small glass containers, each about 1/3 full.
Step 3: Cut the DNA away from the proteins that have been surrounding and protecting the DNA
Add a pinch of enzymes to each test tube and stir gently (so as not to break up the DNA).
In DNA extraction for gel electrophoresis, protein denaturation is accomplished with heat and/or
other chemicals.
In our lab, the chemical papain in meat tenderizer is what separates the DNA from the protein
Step 4: Slowly add alcohol to provide a lighter upper layer for the DNA to move into.
Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the
tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same
amount of alcohol in the tube as pea mixture. DNA is soluble in water. But in alcohol it uncoils and
precipitates leaving behind the other cell components below (that are not soluble in ethanol).
Proteins and fats stay in the bottom pea layer, while DNA floats to the top into the alcohol layer. You
may see them being lifted into the alcohol by bubbles.
You can use a wooden stick or other hook to draw the DNA into the alcohol.
What you have extracted is nucleic acid (DNA and RNA). We cannot use this in gel electrophoresis to
get a DNA fingerprint, because we have extracted all of the DNA (the entire genome). To perform a
gel electrophoresis, you would use restriction enzymes to cut small segments of DNA to be used in
obtaining a DNA fingerprint.