Activity No : DNA Extraction from Apples

Extraction of DNA from any sample (like, plant, animal, blood, tissue or saliva), requires similar basic
principles:
1. The cellular membranes must be disrupted to release the DNA into solution. This is generally accomplished
by adding a detergent. With most tissues, it is helpful to break the sample up into many small pieces (using a
blender) to allow the detergent access to all of the cells instead of just the outermost ones. This step is critical
for extraction from plant tissue where both the cell wall and the cell membrane must be disrupted to gain
access to the DNA.
2. The detergent plus the sample should then be incubated at 55-65°C for 10-15 minutes to accelerate the
disruption of the cell. At this point, the DNA should all be in solution in the liquid.
3. The extra pieces of tissue should be filtered out & the clean liquid (containing the DNA) transferred to a
fresh tube.
4. Depending on how clean the DNA needs to be, there are a number of ways to proceed from this point. The
simplest way to see the DNA is to add an alcohol, such as isopropanol (75-100%) very slowly to form a layer
on top of the filtrate. Because DNA is not soluble in alcohol, it will come out of solution at the interface
between the two layers as white, stringy-looking substance, which can be spooled out of the solution.
[NOTE: Additional methods of isolation all use an alcohol step to precipitate the DNA. Many include clean-
up steps to make the DNA more pure, e.g., using enzymes to digest all of the protein & RNA molecules present
in the sample –such as proteinase K or phenol & then rinsing & re-suspending it in water.]
Extraction of DNA from fruits or vegetables is very similar to extraction from other parts of the plant such as
leaves or stems. First, the tissue needs to be ground to break apart the cells & weaken the cell walls. Then, the
DNA can be extracted.

Objective
In this activity, you will have the opportunity to isolate DNA from apples or other fruit.

Materials& apparatus
Blender Water bath (at 55-60°C) Beaker (500 rnL)
Graduated cylinder (500 mL) Dishwashing liquid (100 mL) Table salt - NaCl (15 g)
Water (450 mL) Isopropyl alcohol / ethanol (ice cold) Funnel filters
Glass rod Apples (2x) Dropper (2ml).

Procedure
1.To 50mL of water, add 7.5 grams of table salt (NaCl) & dissolve. To this mixture, add 50mL of liquid
dishwashing detergent. Mix gently (without foaming) & bring this solution to a final volume of 500mL. This
is the extraction solution.
2. Prepare the hot water bath at 55-60°C. (Optional: prepare an ice bath).
3. Chill sufficient 95-100% isopropyl alcohol in the ice bath or in a freezer. You will need approximately 7mL
for each group conducting the experiment
4. Cut one or two apples into pieces small enough to fit into a blender. Place approximately 500mL of apple
pieces in the blender & cover with enough extraction solution to immerse the whole sample.
5. Blend, pulsing at first, & finally liquifying the apples.
6. Transfer the mixture from the blender to a fresh tube or beaker. Incubate the mixture in a hot water bath for
15 minutes. During this time, stir the mixture continuously to make certain the cells (in the foamy top portion
of the mixture) & the liquid (at the bottom) are heated to 55-60°C. Heating the slurry longer than 15 minutes
will cause the DNA to begin to break down. The heat treatment softens the phospholipids in the cell
membrane & denatures the DNA-degrading enzymes that, if present, would cut the DNA into small fragments
that cannot be extracted.
7. Remove the apple-extraction solution mixture from the water bath and let it cool in an ice bath for 5
minutes, stirring frequently.
8. Filter the mixture through a paper filter placed in a funnel or over a large beaker (a coffee filter can be
used). Try to keep the foam out of the filtrate, which is the location of the DNA. This solution can be stored in
a refrigerator for up to several weeks before proceeding. When the solution is removed from the refrigerator,
warm it to room temperature & swirl to mix.
9. For each person, add 5 mL of the apple filtrate to a glass test tube.
10. Add ice-cold alcohol to the test tube to create an alcohol layer approximately l-2cm thick on top of the
apple filtrate. The best way to accomplish this is to use a dropper. Pour the alcohol slowly down the inside of
the test tube like in the sketch below.

11. Observe for 2 minutes without shaking or mixing. Small bubbles will appear. A cloudiness will be seen at
the interface of the two layers. DNA is not soluble in alcohol. When alcohol is added to the mixture, all of the
components of the mixture, except for DNA, stay in solution, while the DNA at the interface, precipitates out.
12. After 2-3 minutes, insert a glass rod into the solution so that it extends through the interface. Twist & turn
the rod, & gently pass it back & forth between the DNA & the filtrate at the bottom of the tube. Do this slowly
so as not to cause too much mixing in the layers. The DNA should gather on the rod much like thread on a
spool. It will look white & slimy.

For Consideration
1. Where is DNA located in the cell? How would you describe the overall shape of DNA?
2. How would the procedure for isolating DNA from the apple be different from isolating DNA from other
plant or animal tissues?

Reference
Townsley, W. (2001). The Case of Stolen Apples. Forensics for the Biology Laboratory. Burlington,
North Carolina: Carolina Biological Supply Company