The Onion Lab Stock Solutions 1 M Tris pH 8.0 for 100 mL: 12 g Tris (tris-hydroxymethyl aminomethane) Anachemia # AC-9596 VWR/Canlab #9210 85 mL of distilled water adjust pH to 8.0 with HCl add water for a final volume of 100 mL 0.025 M EDTA pH 8.0 for 100 mL: 9.4 g EDTA (ethylene diamine tetra-actetate) VWR/Canlab # 4005 80 mL of distilled water add NaOH pellets to adjust pH to 8.0 add water for a final volume of 100 mL 5 M NaCl for 100 mL 29 g NaCl (sodium Chloride) add water to make 100 mL 10% SDS for 100 mL 10g SDS (sodium dodecyl sulfate) VWR/Canlab B30175-34 add water for a final volume of 100 mL The Onion Lab Introduction The process of isolating DNA from a cell is the first step of 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 and sheared. A “filtrate” is made of onions and treated with a buffer containing “TRIS EDTA” salt and distilled water. The onion due to its low starch content, allows the DNA to be seen more clearly. The TRIS acts as a buffer to maintain a constant pH. EDTA binds up the positive ions to prevent enzymes (DNAses) from chewing up the DNA. The salt shields the negative phosphates of the DNA which allows these ends to come closer so that they can precipitate out of a cold alcohol solution. A detergent (SDS) is added which causes the cell membrane to break down by emulsifying the lipids and proteins of the cell and disrupting the polar interactions that hold the cell membrane together. It also serves to strip the histones from the DNA. Materials onions Test tubes Ice cold ethanol nylon stocking 10 mL pipets 1 M Tris pH 8.0 blender graduated cylinders 0.025 M EDTA pH 8.0 funnel wooden splints 5 M NaCl 10 % SDS PROCEDURE 1. To prepare 100mL of lysing buffer Add to a beaker: 5 mL TRIS 10 mL EDTA 6 mL NaCl 79 mL distilled water 2. Peel and mince 2 onions ( approximately 50 mL of onion) 3. Mix 50 mL lysing buffer and the chopped onion. 4. Homogenize this solution in a blender 45 seconds on low 30 seconds on high 5. Filter the homogenate through a nylon stocking, save the filtrate (liquid coming through) 6. Pour 10 mL of the filtrate into a 50 mL test tubes and distribute to members of the class. 7. To each test tube add 1 mL of SDS (this acts to wash away extra proteins away from DNA). Gently mix without causing foam. 8. add Immediately add 20 mL of ICE COLD ethanol to the test tube by slowly pouring it down the side of the test tube to create a layer on top of the filtrate. (DNA is not soluble in ice cold ethanol. When it is added to the mixture, all the components of the mixture except for DNA, stay in solution, while the DNA precipitates out.) 9. Let the ethanol sit of 2-3 minutes without disturbing it. Bubbles will form and DNA will precipitate out of the solution at the interface. 10. Gently swirl the DNA using a wooden splint. It will look like whitish mucous. 11. The DNA may be lifted out carefully and dried on an absorbent towel. Laboratory Record Sheet 1. Where you surprised by the appearance of the DNA? EXPLAIN why or why not. 2. Name three properties of DNA that are demonstrated in this lab. 3. Do you think that any of the three properties would be useful in identifying DNA? EXPLAIN your answer. 4. How could you be certain that the substance you extracted was DNA? 5. What was accomplished by using a blender to make your homogenate? How about the detergent? Alcohol?