06-02-2013, 09:43 AM
Extracting Onion DNA
Abstract
In this project, you'll learn how to isolate DNA from onion cells, separating it from other cellular components in a manner that still preserves its structure and sequence. In the end, you'll have enough DNA to see with the unaided eye, and you'll be able to spool it to demonstrate its strand-like structure.
Objective
This procedure is designed to extract DNA from onion in sufficient quantity to be seen and spooled.
Introduction
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.
It is both interesting and important to understand the reason for some of the steps in the procedure below. 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.
Materials and Equipment
This experiment is based on the use of household equipment and supplies.
• 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
• light-colored dishwashing liquid or shampoo, such as Dawn® or Suave® Daily Clarifying Shampoo
• large onion
• table salt, either iodized or non-iodized
• (optional) meat tenderizer that contains papain, such as Adolph's
• 1 test tube, preferably with a cap, that contains the onion solution. (A narrow glass container, such as a liqueur glass or clear bud vase can substitute for the test tube.)
• pasteur pipettes or medicine droppers
• 95% ethanol (grain alcohol). (Note: Make sure to purchase 95% ethanol, not methanol. If you can not find it locally, it can be purchased from online suppliers like Carolina Biological, item number 86128. Use caution when handling ethanol, it is very flammable.)
• kitchen timer
• (optional) meat tenderizer and flat toothpicks
Experimental Procedure
1. Prepare two water baths—one at 55-60°C and another filled with ice and water, around 4°C. For the hot water bath, a large metal pot can be used along with a thermometer with an appropriate temperature range. For the ice bath, a mixing bowl filled with ice and water works well.
2. 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.
3. Coarsely chop one large onion with a food processor or blender (may be done by hand if neither is available) 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.
4. Cover chopped onion with the 100 ml of solution from step #2. The liquid detergent causes the cell membrane to break down and dissolves the lipids and proteins of the cell by disrupting the bonds that hold the cell membrane together. The detergent causes lipids and proteins to precipitate out of the solution. NaCl enables nucleic acids to precipitate out of an alcohol solution because it shields the negative phosphate end of DNA, causing the DNA strands to come closer together and coalesce.