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DNA Isolation from Onion

By Kate Dollard




INSTRUCTOR'S GUIDE

INTRODUCTION

Many students find studying DNA difficult because it is so small that the concepts are quite abstract. This lab enables students to work with DNA concretely by easily isolating chromosomal DNA using the same basic tools and methods that scientists use. The lab is a good introduction to using pipets and to using the metric system. If the chemistry of the solutions is taught it is also a great practical application.

OBJECTIVES

  1. Students will apply measurement skills using the metric system.
  2. Students will use knowledge of chemistry to understand the process of isolating DNA from other cell contents.
  3. Students will apply knowledge of cell structure to understand the process of isolating DNA.

MATERIALS

  • 1M TRIS pH7.5-8.0
  • 0.5M EDTA
  • 5M NaCl
  • 2 yellow onions
  • 10% SDS
  • 1 pasteur pipet with the tip bent into a hook for each group
  • 1 large test tube per group
  • 1 test tube rack per group
  • marking pens
  • 1 large funnel
  • cheesecloth
  • 3 250 mL beakers
  • 20 mL ice cold ethanol per group
  • Distilled Water
  • pipet bulbs
  • 1 1 mL pipet per group
  • 1 5 mL pipet per group
  • 1 10 mL pipet per group
  • meat tenderizer


TEACHER PREPARATION

  1. 1M TRIS pH 7.5 (to make 500 ml)
    Trizma base 60.5g
    distilled water 400 mL
    Add HCl until pH7.5 is reached. Add distilled water to 500 mL total volume. This solution can be used immediately as is. If sterile, it will store indefinitely at room temperature. Sterilize by using a filter sterilizer, autoclave or pressure cooker.

  2. 0.5M EDTA (to make 500 mL)
    EDTA 93g
    Distilled water 350 mL
    Adjust to pH8.0 with Sodium Hydroxide (about 25 mL of 10M). The EDTA will not all dissolve until pH8 is reached. Add distilled water to 500ml. This solution can be used immediately as is. If sterile will store indefinitely at room temperature. Sterilize by using a filter sterilizer, autoclave or pressure cooker

  3. 5M Sodium Chloride (to make 500 mL)
    NaCl 145g
    Distilled water Add to NaCl for a total volume of 500 mL
    * It will take awhile to dissolve - try a stir plate or some heat to get all of the salt into solution. Sterilize by using a filter sterilizer, pressure cooker or hotplate. If sterile it will store indefinitely at room temperature.

  4. 10% SDS (to make 500 ml)
    Sodium Dodecyl Sulfate (SDS) 50g
    Distilled Water Add to SDS for a total volume of 500 mL
    *Be careful not to shake or stir too vigorously because too many bubbles will form. Sterilize by using a filter sterilizer, pressure coooker or hotplate. If sterile it will store indefinitely at room temperature.

  5. Meat tenderizer (protease)
    meat tenderizer (ex. Adolphs) 3g
    distilled water 50 mL
    If tenderizer doesn't go completely into solution, then mix well before pipetting.


TEACHER TIPS
  1. Make solutions as shown above. If sterile they will last for a couple of years.

  2. If you want to teach more chemistry, have the students make the Tris, NaCl and SDS. Give them the molecular weights and have them calculate the amounts of chemical needed for a desired volume of solution.

  3. Teach how to use the pipets by having different students volunteer to pipet the solutions in the group preparation. By doubling the recipe, twice as many students will have a chance to pipet. Have a beaker of water for those students who just want to practice.

  4. If you are really pushed for time, or just want to simplify the procedure, make the lysing buffer before lab begins and begin the lab at step three.


ANSWERS TO STUDENT QUESTIONS

  1. Buffer: A solution that will maintain a constant pH, chelate metal ions and generally maintain an environment that will prevent disruption of the experiment.
    Precipitate: The solid which is visible when a substance becomes insoluble from a solution.
    Filter: To separate by passing a mixture through a selective membrane.
    Emulsify: To physically break up lipid into smaller lipid globules.

  2. To maintain an environment in which the DNA will not be degraded and to help separate the DNA from other cell components. Tris maintains a constant pH. EDTA chelates metal ions. NaCl provides Na+ ions that will block negative charge from phosphates on DNA.

  3. Negatively charged phosphates on DNA cause molecules to repel each other. The Na+ ions will form an ionic bond with the negatively charged phosphates on the DNA, neutralizing the negative charges and allowing the DNA molecules to come together.

  4. Any non-starchy vegetable should work. Potatoes would not work well.

  5. Answers will vary.

  6. DNA in cells will absorb UV light. When it does it can be damaged. If the damage occurs in a cell cycle gene, then growth may become uncontrolled leading to skin cancer.



STUDENT PAGE 1

DNA Isolation from Onion

INTRODUCTION The process of isolating DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate the DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up or shredded. The procedure you will be doing is a modification of the "Marmur" preparation which is used worldwide in biotechnology laboratories.

A "Filtrate" is made of onions treated with salt, distilled water and detergent(SDS). An onion is used because it has a low starch content which allows the DNA to be seen more clearly. The salt shields the negative phosphate end of DNA which allows these ends to come closer so they can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to breakdown by emulsifying the lipids and proteins of the cell and disrupting the polar interactions that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution. Collectively, the salt solution and detergent are referred to as a lysing buffer.


PROCEDURE

PART 1:Make 100mL Lysing Buffer* (DONE AS A CLASS) *Enough for 5-8 groups of students

  1. Mix 5mL 1M Tris pH7.5
    5ml .5M EDTA
    6ml 5M NaCl
    84ml Distilled water
  2. Peel and mince 1 small onion---approximatedly 50-75ml of onion.
  3. Mix 100mL buffer and the chopped onion.
  4. Homogenate in a blender for 45 seconds on low speed.
  5. Place four layers of cheesecloth.into the funnel. Place the funnel into a 250ml beaker. Pour the blended onion mixture into the funnel and let sit to filter.


PART 2: Precipitating DNA (Done by each group)

MATERIALS

1 50mL-test tube 1 10mL-pipet
1 mL 10%-SDS 1 5mL-pipet
1 glass stirring rod 1 1mL-pipet
20 mL ice cold ethanol      pipet bulb
methylene blue microscope, slide and coverslip
4 mL of meat tenderizer solution
spectrophotometer and cuvettes (optional)



STUDENT PAGE 2
  1. Pipet 10mL of onion filtrate into the 50ml test tube

  2. Add 1mL of 10% SDS.

  3. Add 4mL of meat tenderizer solution.

  4. Place on ice for 5 minutes

  5. Immediately add 15-20ml of ice cold ethanol to the test tube by SLOWLY pipeting the ethanol down the side of the tube. A clear layer of ethanol should form on top of the onion filtrate. DNA is not soluble in ice cold ethanol. When ethanol is added to the mixture, all the components of the mixture except for DNA stay in solution, while the DNA pecipitates out.

  6. Let the tube sit for 3-5 minutes without disturbing it. Bubbles will form and DNA will predipitate out of solution. The DNA becomes visible as white strings in the ethanol layer. For best results, let sit in the refrigerator overnight.

  7. Spool DNA by snagging it with a pasteur pipet with a hook bent into the tip. To make the hook place the tip of the pipet in a flame. In a few seconds it will be hot enough to bend the tip into a hook by grasping the end with some forceps.

  8. Store DNA on a dish or in a small test tube.

  9. Put a small amount of the DNA on a slide. Add a drop of methylene blue. Put on a coverslip and observe under a microscope.

OPTIONAL:

  1. Dissolve in 5ml of distilled water.

    Determine Purity of DNA sample by spectrophotometry reading at A260 and A280

    (Directions for LKB Spectrophotometer at CRLS)

  2. Turn on the spectrophotometer and let it warm up at least 15 minutes.

  3. Fill one UV cuvette with distilled water. This is your blank.

  4. Fill another UV cuvette with your DNA sample from step 10.

  5. Lift the compartment door on the right and place the blank cuvette in holder 1 and the sample in holder 2.

  6. Adjust the wavelenth for absorbance to 260nm

  7. Push the "cell number" button until cell 1 appears.

  8. Push the "set reference" button. This will substract any absorbance by the distilled water blank away from any sample readings. Therefore any absorbance will be due to nucleic acids and protein in the sample.

  9. Advance to cell 2

  10. Record the reading in the chart on the next page.

  11. Repeat step 15-19 with the wavelength set at 280nm.



STUDENT PAGE 3



A260, A280, ratio A260/A280

Nucleic acids, DNA and RNA, absorb at 260nm and proteins absorb at 280nm. A ratio 260nm/280nm of 1.8-1.9 indicates pure DNA. A ratio of 1.9-2.0 indicates pure RNA. Your sample is a mix of DNA and RNA and protein. The ratio will be low if there is a lot of protein absorbing at 280nm.


QUESTIONS

  1. Vocabulary check. Define each of the following: Buffer

    Precipitate

    Filter

    Emulsify

  2. What is the purpose of the lysing buffer? Describe the role of the different chemicals if possible.






STUDENT PAGE 4
  1. Draw a picture of the negatively charged DNA and the positively charged sodium ions. Why are the Na+ ions necessary for the DNA molecules to come close together?








  2. What other vegetables could be used for this lab?



  3. How pure was your DNA sample? Explain.





  4. Ultraviolet light has a wavelength between 10-7 and 10-9 meters. Given this information and the information about DNA and energy absorbance in this lab, why is too much sun exposure a cause of skin cancer?


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