X
تبلیغات
زیست - نحوه استخراج DNA
مطالب زیست

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 60o C (140o 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 (60o 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

Supplies provided to the class

  • 1 test tube for each student, 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)
  • (optional) meat tenderizer and flat toothpicks
  • laboratory instructions

DNA EXTRACTION FROM ONION
PAGE 2

TEACHER PREPARATION

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

5. Put the measuring cup in a hot water bath at 55-60o 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.)

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 test tubes, one for each student. 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.

DNA EXTRACTION FROM ONION

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

  1. (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.

  1. 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.

  1. 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.

Prepared by the Office of Biotechnology, Iowa State University

revised 3/94

Dr. Walter R. Fehr
Director of Biotechnology
1210 Molecular Biology Building
Ames, IA 50011
ph: 515/294-6865 FAX: 515/294-4629 email: W_Fehr@Molebio.iastate.edu

 

+ نوشته شده در  دوشنبه پانزدهم بهمن 1386ساعت 13:18  توسط دوست  |