Pre-Lab Exercises Lab 8: Biochemistry Name Date Section 1. List the 3 basic components of a DNA nucleotide, and draw a simple picture to show how they interact. 2. Consider the amine bases in DNA. List the 4 bases. Why do bases only bond with certain other bases? How do the bases bond with each other? 3. List 4 differences between DNA and RNA. Page 1 of 5
Chemistry 100 Lab 8: Biochemistry DISCUSSION (NOTE: This lab was adapted from Laboratory Experiments for General, Organic and Biochemistry, 6 th ed., Bettelheim/Landesberg) Most cells in our body contain DNA, the molecule that makes up the genes that control our physical characteristics. Half your DNA came from your mother, and half from your father. Your DNA is what determines your eye color, hair color, height, blood type, and all other physical properties of your body. In our cells, DNA is found inside the nucleus, wrapped around basic protein molecules called histones (kind of like thread wrapped around a spool). This combination of DNA and protein is called a nucleosome. The DNA does not leave the nucleus, so when new proteins or other structures need to be made, RNA acts as a messenger to help translate the DNA and carry these instructions to the cell s machinery that will go to work. RNA, while it can come inside the nucleus when needed, is found primarily outside the nucleus in organelles and in the cytoplasm. In this experiment, we will isolate DNA from onion cells. First, we will break up the cells in order to release the DNA. This will be done both mechanically (grinding the cells with sand) and chemically (adding a detergent ). The chemical step is particularly important because the detergent not only helps break down the cell membranes so the DNA can flow out, it also helps inactivate the cells DNA-degrading enzymes (nucleases) that would otherwise break down the DNA into its component nucleotides. Second, after the DNA is in solution, we must separate it from the rest of the cellular components. Proteins and water-soluble molecules will stick to the DNA until we add a sodium chloride solution this will minimize the interaction between positively-charged protein molecules and negatively-charged DNA. The addition of ethanol then separates the large molecules (DNA being the largest of the molecules in solution) from the smaller molecules (protein and RNA). The DNA will form a thread-like precipitate that can be spooled onto a rod, while the gelatinous mixture of proteins and RNA are left behind. This crude extract of DNA will still contain some contaminant proteins, but it is mostly DNA. Finally, we will confirm that our extract truly contains DNA by using a diphenylamine test. The appearance of a blue color is specific to DNA and will show that our extract is indeed that specific nucleic acid. PROCEDURES Releasing the DNA 1. Cool a mortar in ice water. Add 20 to 30 g of diced yellow onion and 20g of acid-washed sand. Grind the onion and the sand vigorously with a pestle for 5 minutes to disrupt the cells. A yellowish mush will form. 2. Using a hot plate, warm 100 ml distilled water to 60 C. Add 10g NaCl and add 10 ml of detergent. Stir to dissolve. 3. Add the ground onion and sand mush to 30 ml of the saline-detergent solution. Using a glass rod, stir the solution for 5 minutes, all the while maintaining the temperature of the solution at approximately 60 C. Page 2 of 5
Separating the DNA 1. Support a funnel with a ring clamp on a ring stand above a 150-mL beaker. Add cheese cloth to the funnel and decant the cell solution onto the cheese cloth. Let the yellowish liquid filter into the beaker, while gently squeezing the cheese cloth to obtain the maximum yield, leaving the cellular debris and sand behind. Cool the solution to room temperature. 2. To the viscous DNA-containing aqueous solution, slowly add twice its volume of cold absolute ethanol, taking care that when you add the alcohol, it flows along the side of the beaker, settling on top of the aqueous solution. 3. THIS STEP IS CTIRITAL! Insert a flame-sterilized glass rod gently into the solution, reaching just below the interface of the DNA-alcohol solution. This is where the DNA will form a thread-like precipitate. Rotate (do not stir) the glass rod in one direction to spool the DNA precipitate onto the glass rod. 4. Transfer the spooled DNA on the rod into a test tube containing 95% ethanol. 5. Discard the alcohol/detergent solution remaining in the beaker into specially labeled waste jars (do not pour down the sink). Confirming the DNA 1. Remove the rod and the spooled DNA from the test tube. Dry the DNA with a clean filter paper. Describe the appearance of the crude DNA. 2. Dissolve the isolated crude DNA into a test tube containing 2 ml citrate buffer. 3. Set up a beaker of distilled water and bring it to a boil on a hot plate. 4. Set up 3 more dry and clean test tubes. Into the test tubes, add 2 ml of each of the following: Test Tube Solution 1 1% glucose 2 1% ribose 3 1% deoxyribose 4 Crude DNA extract (from step 2) 5. Add 5 ml diphenylamine reagent to each test tube. Mix the contents of the test tubes. Heat the test tubes in the beaker of boiling water for 10 minutes. Note the color. Page 3 of 5
Lab Station Check Out # REPORT Name Lab Partner Date Section Confirming the DNA 1. Describe the appearance of your crude DNA extract after spooling. Diphenylamine Test Solution Color 1% glucose 1% ribose 1% deoxyribose Crude DNA extract Did the diphenylamine test confirm the presence of DNA in your extract? Post-Lab Questions 1. Could you do without using the detergent in the procedure? Explain your answer. Page 4 of 5
2. In the diphenylamine test, you have both a ribose solution and a deoxyribose solution. a. What are these 2 chemicals? b. What is the main difference between the 2? c. Draw the structure of each. i. Ribose ii. Deoxyribose 3. Is the diphenylamine reagent able to distinguish between ribose and deoxyribose? Could it also be used to distinguish between DNA and RNA? 4. DNA, RNA and proteins are all relatively large molecules. However, DNA can be isolated by the spooling procedure, while the other molecules are left behind. Why does this work for DNA and not the other molecules? 5. Draw a simple DNA molecule, containing at least 3 nucleotides on each DNA strand, and show how the 2 strands are bonded together. Be sure to pair the correct bases! Page 5 of 5