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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. DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction: 1. Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methodsblending, grinding orsonicating the sample. 2. Removing membrane lipids by adding a detergent or surfactants. 3. Removing proteins by adding a protease (optional) 4. Removing RNA by adding an RNase (often done). 5. Precipitating the DNA with an alcohol usually icecold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt. Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+, which prevents enzymes like DNase from degrading the DNA. Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation. If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.

Principle -

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. DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.

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 60C (140F), 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 (60C)(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 bathsone at 55-60C and another filled with ice and water, around 4C. 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. 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. 5. Put the measuring cup in a hot water bath at 55-60C 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.) If using a large metal pot for water bath, remove the pot from the stove before placing the onion-containing measuring cup insidethe procedure is safer if the pot is off the burner. Continue to monitor temperature of water bath and make adjustments as needed (i.e., adding hot or cold water). The heat treatment softens the phospholipids in the cell membrane and denatures the DNAse enzymes which, if present, would cut the DNA into small fragments so that it could not be extracted. 6. 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. 7. 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 you filter the onion mixture, try to keep the foam from getting into the filtrate. It sometimes filters slowly, so you might want to put the whole set up in the refrigerator and let it filter overnight. 8. Dispense the onion solution into a test tube. The test tube should contain about 1 teaspoon of solution or be about 1/3 full. For most uniform results among test tubes, stir the solution frequently when dispensing it into the tubes. There is not an advantage to dispensing more than one teaspoon of solution into a test tube. The solution can be stored in a refrigerator for about a day before it is used for the laboratory exercise. When the solution is removed from the refrigerator, it should be gently mixed before the test tubes are filled. 9. (Optional) Add two toothpicks full of meat tenderizer to the onion solution, cap the tube, and mix gently to avoid foaming. Meat tenderizer contains papain, an enzyme that will clean extra proteins away from DNA. 10. 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) Or, put about 1 cm of alcohol into the bottom of a test tube and add the onion solution. (c) Or, 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. 11. 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. A wooden skewer or nut pick (small metal rod with curved tip) may also work well for spooling DNA if Pasteur pipette is unavailable. DNA has the appearance of white mucus.

Observation Bibliography
Rice, M. (n.d.). DNA Extraction Principles: How do you get DNA out of a whole organism? Retrieved June 30, 2010, fromhttp://www.slideshare.net/meganrice/dna-extraction-principles

This project is from the "Understanding Genetics: Human Health and the Genome" exhibit at The Tech Museum of Innovation in San Jose, CA: http://www.thetech.org/genetics/medicine.php

Another similar project can be found from the Santa Clara College Biotechnology Education Program (SCCBEP) at San Jose State University: SCCBEP, 2002. "DNA Spooling from onions," Santa Clara College Biotechnology Education Program (SCCBEP), San Jose State University, CA. [accessed March 6, 2007] http://www.science.sjsu.edu/sccbep/resources/curriculum/dna_spooling_strawberry.pdf

"Understanding Genetics: Human Health and the Genome" exhibit at the Tech Museum of Innovation and click on the "Zooming Into DNA" online exhibitRosa, C. et. al., 2007. "Zooming Into DNA," The Tech Museum of Innovation, San Jose, CA. [accessed March 6, 2007]http://www.thetech.org/genetics/index.php