Extraction of DNA from Onion Jo Ann Lane 1994 Woodrow Wilson Collection
Overview To most students of biology, DNA is an abstraction. You can memorize the names and structures of the nitrogenous bases and know k now all about the history of DNA's discovery, but until you actually handle DNA, it remains a strange and mysterious substance. The purpose of this laboratory is to give you firsthand experience with DNA by isolating it from plant tissue. You will start with whole onions and end with a relatively pure preparation of DNA, containing literally millions of genes. Once isolated, the DNA can be stored in alcohol or dried out. It will actually be possible for you to hold in your hands the key to an organism's development and structure.
Objectives 1. To become familia familiarr with the physical physical properti properties es of DNA by isolating isolating it from from living living tissue tissue 2. To learn the the purpose of of each step step in the isolati isolation on procedure procedure as it relate relatess to the physical physical and biochemical characteristics of the genetic material
Materials and Equipment The following materials can be shared by a group of students: •
blender
•
60û C water bath
•
thermometer
•
ice bucket
• •
balance (0.1 g scale) 95% ethanol kept on ice
The following materials are needed for each student or pair: •
plastic gloves
•
100 ml homogenization medium
•
cutting board
•
medium onion
•
knife
•
weighing boat
•
funnel
DNA Isolation Procedure
cheesecloth (4 thicknesses 2-100 ml graduated cylinders (one kept on ice) 250 ml beaker 500 ml beaker 1000 ml beaker glass stirring rod
Homogenization: Before DNA can be released from the nuclei of the onion tissues, the cell walls, plasma membranes, and the nuclear material must first be broken down. This step can be accomplished by homogenizing the onion tissues in a blender. The detergent solution causes the cell membrane to break down and emulsifies the lipids and proteins of the cell by disrupting the polar interactions that hold the cell membrane together. The DNA can then be separated from the chromosomal proteins by the chemical components of the homogenizing medium which will cause the proteins to precipitate out of solution. 1. Wearing Wearing plastic plastic gloves, gloves, dice a medium-si medium-sized zed onion into into cubes no no larger larger than 3 mm. (This (This step may have already been done for you to save time.) Plastic gloves prevent DNAse enzymes on your hands from cutting the DNA into small fragments so that it will not spool. 2. Weigh out 50 g of diced diced onion. Transfer Transfer all all of the weighed weighed materi material al to a 250 ml beaker. beaker. 3. Add 100 ml ml of homogeniz homogenizing ing medium medium to the the diced diced onion and and incubate incubate the beaker beaker in in a 60û C water bath for 15 minutes (no longer!). This heat treatment softens the onion tissue and allows penetration of the homogenization solution. It also denatures many enzymes that could interfere with the isolation procedure. 4. Quickly Quickly cool your your preparation preparation to 15-20û 15-20û C in an ice ice bath (a slush slush of ice ice and water). water). This step should be accomplished in about 6 minutes and prevents the denaturation of DNA. 5. Pour your your cooled cooled preparation preparation into into a blender blender and fasten fasten the lid. lid. Homogenize Homogenize for 45 seconds at low speed, followed by 30 seconds at high speed. Homogenization breaks open the cells and releases their contents (carbohydrates, proteins, fats, and nucleic acids). 6. Pour the the homogenate homogenate from the the blender blender into a 1000 ml ml beaker. beaker. Allow it it to stand stand in an ice bath for 15-20 minutes. 7. Filter Filter the homogenate homogenate through through four four thicknesse thicknessess of cheeseclot cheesecloth h into a 500 ml beaker, beaker, taking care to leave the foam behind.
Precipitation of DNA: The homogenate should contain only DNA and the components of the homogenizing medium. Of the components remaining in the homogenate, only DNA is not soluble in ice-cold ethanol. Therefore, when ice-cold ethanol is added to the homogenate, all the components of the homogenizing medium stay in solution-except DNA. If the instructions have been followed carefully so that the molecular structure of DNA remains intact, the genetic substance should precipitate p recipitate as a thick, stringy, white mass that may be spooled out by winding it on a glass rod. If the DNA has been damaged, it will still precipitate, but as a white, fuzzy mass that cannot be collected on a glass rod. 8. Place your your beaker beaker with its its filtered filtered homogenat homogenatee into an ice bath. bath. Let it cool until until it reaches reaches 10-15û C (about 10-15 minutes). 9. Measure Measure out 80 ml of ice-col ice-cold d ethanol into into a cold graduat graduated ed cylinder. cylinder. Slowly Slowly add add the ethanol down the side of your beaker until the white, stringy DNA precipitate appears. It may not take all 80 ml of the alcohol to precipitate your DNA.
10. Spool out, or wind up, the stringy DNA onto a glass glass rod by rotating rotating the rod in one direction only in the beaker of DNA. Continue to rotate the rod as you move it in large circles through the beaker. 11. If you want to keep your DNA, gently ease it off the end of the glass rod into a vial filled filled with 50% ethanol. Be sure the cap is tight enough to prevent leakage.
Questions 1. Why is it it important important to rotate rotate the the rod in the same same direction direction when when spooling spooling the DNA onto onto the rod? 2. What did did you learn learn about the propert properties ies of DNA DNA during this this laborato laboratory ry period? period? 3. What structu structural ral character characteristi istics cs of DNA allows allows it to be spooled spooled out on a glass glass rod? Why Why is it not possible to spool out precipitated proteins? (Hint: Compare the relative lengths of DNA and protein molecules.)
Teacher Information 1. Prepar Preparati ation on of homog homogeni enizat zation ion mediu medium: m: sodium laural sulfate (SDS or SLS)
50.000 g
sodium chloride
8.770 g
sodium citrate
4.410 g
ethylenediamine tetraacetic tetraacetic acid (EDTA) 0.292 g 2. Add distille distilled d water to make make 1 liter liter of solution. solution. Do not place place in refrige refrigerator rator or the SDS will turn the solution an opaque white color. If the homogenization medium gets cold at any time, it will turn white, but this will not affect its function. 3. Ethanol Ethanol must be cold cold for this this procedure procedure to work. work. Place Place a bottle of ethanol ethanol and one graduated cylinder for each lab group in a freezer overnight. Be sure the cap is loose and the bottle is not completely full. Place the bo ttle on paper towels. It will not freeze. Just before lab dispense the ethanol into smaller containers and put on ice. Or you can fill one smaller bottle for each lab group and pass out the bottles and graduated cylinders directly from the freezer or from a cooler filled with ice just before the students will use them. 4. Have the students students wear wear gloves gloves and tell tell them not to to touch the inside inside of contai containers ners because because DNAse enzymes from their hands will break the DNA into small fragments so that it will not spool at the end of the lab. Rinse all glassware with distilled water. 5. Stress Stress with the the students students that they must must follow follow the directions directions careful carefully ly since the the temperatures and timings are crucial to the procedure. 6. Scoring Scoring the end of the the glass rods rods with with sandpaper sandpaper will will help the DNA DNA adhere to the the rod while spooling. Do not touch the end of the rod with your fingers. 7. You may have have some small small vials vials and 50% ethanol ethanol availabl availablee so that the student studentss may save save their DNA.
8. To save time time you may use use a blender blender to chop chop up the onions onions and and dispense dispense them them in 50 g portions. 9. There is a lot lot of "waiting "waiting time" time" in this this lab, so a workshee worksheett on DNA can be complet completed ed during this time. 10. Wear a mask when massing the the sodium laural sulfate-it sulfate-it is very powdery and gets into the air.
