Chapter 21 - Lab Exercise Perform the following Experiment. Answer the questions posted at the end of the experiment.

Chapter 21 - Lab Exercise

Perform the following Experiment. Answer the questions posted at the end of the experiment. Submit your answers as a WORD document attachment to a posting in the drop box for this unit.

More experiments:

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How to Extract DNA From Fruits

G. Carboni, January 2007

Text editing by Donald Desaulniers, Ph.D.

Figure 1 - Test tube with DNA extract

CONTENTS

INTRODUCTION

PROCEDURE

Summary of the procedure

Preliminary operations

Preparation of the extracting solution

Preparation of the mush

Extracting the DNA

Filtration

Removing the proteins

Precipitating the DNA

Observation through the microscope

CONCLUSION

REFERENCES

INTRODUCTION

In recent years, it is not uncommon to read articles on DNA in both scientific and popular magazines. DNA is regularly mentioned in the news and is often featured in TV detective or crime-scene investigation dramas. DNA, also known as DeoxyriboNucleic Acid, is a long molecule that holds the genetic information for all living beings, be it vegetable, animal or a simple microorganisms. It is capable of copying itself and can synthesize RNA (RiboNucleic Acid). In more evolved or complex forms of life, DNA is contained in the nucleus of the cells. Except for the red blood cells of mammalians, which are devoid of a nuclei, all cells of a living being have their own DNA. The cells of an organism use certain parts of the DNA molecule, or genes, to produce the proteins they need to function. For a more detailed description of DNA including its structure, its functions and the mechanism by which proteins are produced, the reader should consult the texts listed [1] the Reference section of this paper, which are well written and contain excellent illustrations. In this article, I describe a simple experiment that will allow you to extract a bit of DNA from a banana, however, you can also try it using other fruits and even vegetables. It is an experiment that can be performed both at home and in a school laboratory

PROCEDURE 

SUMMARY OF THE PROCEDURE

The procedure described below exploits the fact that the external membrane of cells and that of their nuclei are composed of fatty substances that can be broken down using a simple detergent. The first operation in this procedure is to break-up the fruit into a pulp or mush so that the cells are separated each from other as much as possible thereby exposing them to the action of the detergent. Secondly, the detergent is added to the pulp of the fruit so as to release the DNA from the cell membranes, which encapsulate it. Thirdly, it is necessary to filter the mixture to separate the nucleic acid from the remains of the cellular membranes. Finally, the DNA is precipitated in alcohol where it becomes visible. The DNA you obtain using this procedure can be observed with a microscope and can be used for other experiments like electrophoresis or other experiments.

PRELIMINARY OPERATIONS 

MATERIALS

- pot;

- thermometer; 

- plastic salad bowl; 

- ice cubes;

- 50 cc of 70 - 95 % 70-95% isopropyl or denatured alcohol (ethanol) in a closed bottle;

- rags and absorbent paper tissues.

METHOD

- The day before the experiment, prepare some ice cubes;

- at least 2 hours before, place in a freezer a sealed vapor-tight plastic bottle with 50 cc of 70-95% isopropyl or ethyl denatured alcohol. This container must to be closed tightly to prevent alcohol vapors from being released since they are flammable;

- 15 minutes before starting the procedure, warm a pot of tap water to 60°C;  

Figure 2 - Before starting the experiment, it is important to perform the preliminary operations described here.

Figure 3 - Preparation of the extracting solution (Distilled 

water, table salt, detergent, syringe, 100 cc beaker and spoon).

PREPARATION OF THE EXTRACTION SOLUTION 

As mentioned previously, the DNA is held inside the nucleus of the cells of the fruit we are using. To free the DNA, it will be necessary to breakdown the membranes of the cells as well as those of the nuclei. As these membranes are made up of phospholipids, which are molecules rich in fats, we will dissolve them by using a simple household detergent. We will also use a little table salt, which helps to eliminate the proteins, called histones, on which the DNA is wrapped.

MATERIALS

- 100 cc of distilled water but tap water can also be used ;

- a scale to weigh few grams (if possible);

- 3 g of table salt (a half teaspoon);

- 10 cc of liquid detergent;

- a 10 cc syringe without needle;

- a 100 cc beaker;

- a glass rod.

METHOD

- Pour 3 g of salt and 80 cc of distilled water in a 100 cc beaker;

- mix until the salt is completely dissolved;

- with the syringe, take 10 cc of liquid detergent and add it to the solution;

- add distilled water until you obtain a total volume of 100 cc;

- while avoiding to produce bubbles, mix to homogenize the solution;

- the extracting solution is ready.

PREPARATION OF THE PULP 

This operation serves to separate the cells from each other and to better expose them to the action of the extraction solution.

MATERIALS

- 100 g of banana (or: kiwi, apple, pear, kaki, peas, onion, etc.);

- balance;

- knife;

- chopping board and fork;

- 250 cc beaker;

- a teaspoon.

METHOD

- Place 100 g of banana (without peel) on a chopping board and crush it with a fork until you obtain a pulp. If you use an onion, with a knife obtain cubes of about 5 mm of side or less. You can also use a mortar or a blender. If so, do not shred the pulp too much;

- pour the mush in a 250 cc beaker.

Figure 4 - Preparation of the fruit pulp.

EXTRACTING THE DNA 

The aim of this operation is to breakdown the membranes of the cells and their nuclei to free the DNA. The pulp will heated to 60°C to speed up and help the process of breaking down the membrane walls. Heating the pulp also helps to deactivate certain enzymes like DNase that can degrade the DNA. However, if the pulp is held at an elevated temperatures for too long a time the DNA may begin to fragment du to the heat exposure. For this reason it is advised to cool the pulp after approximately 15 minutes in a bath of chilled water. 

Figure 5 - Pour the extraction solution in the pulp and mix.

Figure 6 - The pulp should be kept at 60°C for no more than 

15 minutes and then chilled to about 0°C for 5 minutes.

MATERIALS

- thermometer;

- pot with water at 60°C;

- salad bowl with tap water and ice cubes.

METHOD

- Pour the extracting solution on the mush;

- place the beaker in a bain-marie in the pot with water at 60°C;

- mix the mush so to distribute the extracting solution and to make the temperature uniform;

- after 15 minutes, place the beaker in a bain-marie in the water with ice cubes;

- mix the mush to make the temperature uniform;

- after 5 minutes, remove the beaker from the cold water and prepare yourself for the filtration. 

FILTRATION 

The filtration process is used to collect the liquid rich in DNA and to separate it from the cellular remnants and the other tissues of the fruit, which will be discarded.

MATERIALS

- sieve with a diameter of about 12 cm;

- coffee filter paper (laboratory filter is too much thick). Kitchen absorbent tissue paper can also be used, provided that it does not have any visible holes;

- bowl.

METHOD

- place the sieve on the bowl;

- take a filter paper, soak it and place it in the sieve;

- pour a little pulp on the filter, taking care that is goes through the filter paper ;

- mix with care to help the filtration and avoid ripping the filter paper;

- the filtered liquid you will obtain is rich in DNA.

Figure 7 - Filtering the pulp using a sieve, filter paper and a bowl.

REMOVING THE PROTEINS (optional) 

With this additional operation it is possible to obtain a purer DNA extract, but it it is not essential if you want to observe the DNA. Because DNA is wrapped on proteins called histones is will be necessary to remove these proteins to obtain a DNA extract of higher purity. To remove these histones, you can use proteolytic enzymes like Protease. While you can purchase protease in a shop that sells chemical products, you can also substitute it with a substance that is much easier to find; it is found in the juice of the pineapple which that contains Bromelain, a substance able to breakdown proteins into the amino acids of which they are composed. 

Figure 8 - Obtaining the pineapple juice.

Figure 9 - In a tube, pour 5 cc of filtrate and 1 cc of pineapple juice.

MATERIALS

- Proteolytic enzyme (ex: Protease or pineapple juice); 

- a 5 cc syringe without needle.

METHOD

- In a tube, pour 5 cc of the filtered solution;

- add 1 cc of pineapple juice and mix;

- wait 2 - 3 minutes to let to the bromelain react.

PRECIPITATING THE DNA 

DNA is quite soluble in water and invisible, while it is insoluble in alcohol wherein it precipitates and becomes visible. By adding alcohol to the DNA filtrate solution in the tube, the DNA is rendered visible.  

Figure 10 - Very slowly, pour some ice cold alcohol into

the tube and avoid mixing the alcohol with the filtrate.

Figure 11 - Tube with DNA of the banana mixed with a numerous tiny air bubbles freed from the alcohol which is warming up. In Figure 1, there are less bubbles and the DNA is observed as a milky substance.

MATERIALS

- Some tubes for the possible repetition of the operation;

- tubes holder;

- icy alcohol (kept in a freezer).

METHOD

- Slowly, pour in the tube of the previous step some icy alcohol by avoiding it mix with the filtrate;

- the volume of the alcohol has to be about the same of that of the solution;

- let the tube rest for 5 minutes to allow to the DNA to precipitate and accumulate in the tube.

Now, at the interface between alcohol and the filtrate you should be able to see a milky substance, which tends to increase in volume as time progresses. This milky substance is the DNA of the banana. Unfortunately, inside this milky little mass, you may observe numerous tiny bubbles. The presence of these bubbles is due to the property that the solubility of gases in a cold liquid is higher than in a warm one. While alcohol was in the freezer it likely absorbed some gases that are expelled as the liquid is warmed up. 

OBSERVATION THROUGH THE MICROSCOPE (optional) 

MATERIALS

- some clean microscope slides;

- hook made with a long metal wire;

- dye to stain the nucleus (ex: Toluidine, Methylene Blue, Aceto-Orcein);

- dropper;

- microscope.

METHOD

- with a long metal wire ending with a hook, extract a sample of DNA from the tube and place it on a clean microscope slide;

- level the mass on the slide and stain it with a nuclear dye;

- if necessary, add a little water and mount the coverslip.

By observing this preparation under the microscope, do not expect to see the well-known double helix ladder structure of the DNA. You cannot see it even with an electronic microscope. What you will see are clumps or flocks of DNA material which look like a tangled mass of protein strands as illustrated Figure 12.

Figure 12 - Sample of banana DNA at about 100 X

(stained with a 1% solution of Toluidine).

CONCLUSION 

This experiment was not so difficult to carry out after all, was it not?. The aim of this simple experiment was to provide you with an introduction to the procedures that are used in molecular biology. Often, the techniques used in modern microbiology laboratories are based on simple operations like this one. In other cases the procedures can be quite complex and may involve more sophisticated manipulations and equipment. In all cases a sound knowledge of biology and chemistry is essential to understanding how DNA is used in the fields of life sciences and health sciences. If this experiment has sparked an interest in pursuing future explorations, remember that resources available through the Internet you can lead you to new areas of discovery. If you would like to learn more, look at the document [2] in the References section of this paper. The extraction of the DNA is the first step of many other fascinating experiments.

BIBLIOGRAPHY 

1. Helena Curtis, N. Sue Barnes; "Biology;" Worth Publishers, Inc., New York; A biology text for high schools.
2. http://www.funsci.com/texts/wsites_en.htm (Links to an external site.)
3.   Look for the term: "SAPS". You will find the directions to made other interesting and fun experiments in biology.

Internet keywords: DNA extraction, DNA proteins amino acids ribosome.

1.     What type of organisms and cells contain DNA? 

2.     What is the role of DNA in organisms?

3.     Why would you want to extract the DNA from the organism?

4.     In the procedure, why is the banana crushed or ground up into a pulp?

5.     Why is detergent added to the pulp?

6.  Look up the meaning of the term "bain-marie" (I have never heard of it either.) What is a bain-marie? Give a reference.

7.  What is a good natural source of protease?

8.  What does a protease do?

9.  Compare the solubility of DNA in water and in alcohol.

10.  What is the difference between a filtrate and a precipitate? If you don't know, look it up. You need to know in order to perform the experiment properly.

11.  Macroscopically, what did your DNA look like? Was it pink as in the lab write-up? Try to pull some out with a stick or a tweezers. What's it like?

12.  (optional)The microscopic part is optional because most students don't have a microscope at home. If you did do microscopic part, what did you observe?

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