DNA EXTRACTION FROM ONION

Prepared by the Office of Biotechnology, Iowa State University

CONTENTS

INTRODUCTION

DNA is present in the cells of all living organisms. This procedure is designed to extract DNA from onion in sufficient quantity to be seen and spooled. It is based on the use of household equipment and supplies.

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MATERIALS

For teacher preparation

two 4-cup measuring cups (1000 ml) with ml markings

one 1-cup measuring cup (250 ml) with ml markings

measuring spoons

sharp knife for cutting onion

large spoon for mixing

food processor or blender

thermometer that will measure 60� C (140� F), such as a candy

thermometer

strainer or funnel that will fit in a 4-cup measuring cup

#6 coffee filter or cheese cloth

hot tap water bath (60� C)(a 3-quart saucepan works well to hold the water)

ice water bath (a large mixing bowl works well)

distilled water

clear-colored shampoo, such as Suave Daily Clarifying Shampoo

large onion

table salt, either iodized or non-iodized

(optional) meat tenderizer that contains papain, such as Adolph's

Supplies provided to the class

1 test tube for each student, preferably with a cap, that contains the onion solution. (A narrow glass container or clear bud vase can substitute for the test tube.)

pasteur pipettes or medicine droppers

95% ethanol (grain alcohol)

(optional) meat tenderizer and flat toothpicks

laboratory instructions

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TEACHER PREPARATION

Set up hot water bath at 55-60� C and an ice water bath.



For each onion, make a solution consisting of one tablespoon (10 ml) of liquid dishwashing detergent or shampoo and one level 1/4 teaspoon (1.5 g) of table salt. Put in a 1-cup measuring cup (250 ml beaker). Add distilled water to make a final volume of 100 ml. Dissolve the salt by stirring slowly to avoid foaming.



Coarsely chop one large onion with a knife and put into a 4-cup measuring cup (1000 ml). For best results, do not chop the onion too finely. The size of the pieces should be like those used in making spaghetti. It is better to have the pieces too large than too small.



Cover chopped onion with the 100 ml of solution from step 2.

The detergent dissolves the fatty molecules that hold the cell membranes together, which releases the DNA into the solution. The detergent, combined with the heat treatment used in step 5, causes lipids (fatty molecules) and proteins to precipitate out of the solution, leaving the DNA. The salt enables the DNA strands to come together.



Put the measuring cup in a hot water bath at 55-60� C for 10-12 minutes. During this time, press the chopped onion mixture against the side of the measuring cup with the back of the spoon. Do not keep the mixture in the hot water bath for more than 15 minutes because the DNA will begin to break down.



Cool the mixture in an ice water bath for 5 minutes. During this time, press the chopped onion mixture against the side of the measuring cup with the back of the spoon. This step slows the breakdown of DNA.



Filter the mixture through a #6 coffee filter or four layers of cheese cloth placed in a strainer over a 4-cup measuring cup. When pouring the mixture into the strainer, avoid letting foam get into the measuring cup. It can take more than an hour to recover most of the liquid. The filtering can be done in a refrigerator overnight.



Dispense the onion solution into test tubes, one for each student. The test tube should contain about 1 teaspoon of solution or be about 1/3 full, whichever is less. For most uniform results among test tubes, stir the solution frequently when dispensing it into the tubes. The solution can be stored in a refrigerator for about a day before it is poured into the test tubes. When the solution is removed from the refrigerator, it should be gently mixed before the test tubes are filled.

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STUDENT INSTRUCTIONS

The process of extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up.



Your teacher has already prepared a solution for you, made of onion treated with salt, distilled water and dishwashing detergent or shampoo. An onion is used because it has a low starch content, which allows the DNA to be seen clearly. The salt shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution.

PROCEDURE

Add cold alcohol to the test tube to create an alcohol layer on top of about 1 cm. For best results, the alcohol should be as cold as possible. The alcohol can be added to the solution in at least three ways. (a) Fill a pasteur pipette with alcohol, put it to bottom of the test tube, and release the alcohol. (b) Put about 1 cm of alcohol into the bottom of a test tube and add the onion solution. (c) Slowly pour the alcohol down the inside of the test tube with a pasteur pipette or medicine dropper. DNA is not soluble in alcohol. When alcohol is added to the mixture, all the components of the mixture, except for DNA, stay in solution while the DNA precipitates out into the alcohol layer.



Let the solution sit for 2-3 minutes without disturbing it. It is important not to shake the test tube. You can watch the white DNA precipitate out into the alcohol layer. When good results are obtained, there will be enough DNA to spool on to a glass rod, a pasteur pipette that has been heated at the tip to form a hook, or similar device. DNA has the appearance of white mucus.

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Prepared by the Office of Biotechnology, Iowa State University

revised 1/96