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DNA Spooling

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University of Pittsburgh at Bradford Science In Motion Biology Lab 011DNA Spooling: Isolating Banana DNAIntroduction:DNA molecules are long, slender molecules that carry the heritable information of organisms on to future generations. Because of their size, it is impossible to see a single DNA molecule with the naked eye. It would take about 300,000 DNA molecules side by side to make a bundle as thick as a human hair. When subjected to certain conditions, it is possible to collect “large” amounts of DNA to make it visible. This process of collecting DNA is referred to as spooling. During spooling, a buffer solution made from soap and salt (NaCl) is mixed with the cells. The soap from the buffer solution disrupts the cell membrane’s phospholipid bilayer by reacting with the phosphate group of the phopholipid. This releases the cellular components into the buffer. Once the components are released, the NaCl in the buffer binds toseveral of the negatively charged phosphate groups of the DNA’s sugar-phosphate “backbone,” shielding some of the negative charge of the DNA. Because some of the negative charges are shielded, the DNA molecules can loosely bind together. After mixing the buffer with the DNA source, the buffer/DNA solution is carefully overlaid with isopropanol. In the presence of salt, RNA and DNA precipitate from solutions containing high percentages of ethanol or isopropanol. Due to its size and abundance, chromosomal DNA forms viscous, clotted masses during alcohol precipitation. A plastic loop is used to mix the two liquids at their interface and collects the DNA as it precipitates from solution at the mixing zone. Small fragments of DNA and degraded RNA usually contaminate the chromosomal DNA during extraction procedures. They are also precipitated by the alcohol, but have little tendency to spool on the loop because they are too short and form finer, more uniform precipitates. Spooling can consequently be viewed as a method that partially purifies and concentrates high molecular weight DNA.The purification of chromosomal DNA is frequently the first step in molecular cloning experiments. The precipitate can be collected and redissolved in a smaller volume. This is a convenient way to concentrate nucleic acids. Alcohol precipitations also remove small molecules, such as buffer salts, sugars and amino acids from nucleic acid precipitations since they remain in solution.Objective:1. Students will extract and isolate DNA from a banana after first making a buffer solution and by following common DNA spooling techniques.2. Students will be able to explain how a buffer solutions disrupts the plasma membrane releasing cellular components into the solution.3. This experiment will provide a “hands-on” activity and opportunity for students to develop an appreciation for the physical nature of DNA and the process of DNA purification.Inquiry Extension:Allow students time to study DNA and DNA spooling through experimentation with other sources of DNA. 1Safety:Wear gloves and goggles as standard laboratory practice.Materials:sodium chloridesodium bicarbonatedetergenttap water50 ml and 100 ml beaker ice cold isopropanolbanana forkfunnelfiltertest tubeplastic loopMethylene Blue Plus DNA stainWax PaperProcedure:1. Buffer Preparation: Mix the following in a clean 100 ml beakera. 60 ml tap water b. 0.75 g sodium chloride – 1/8 tsp.c. 2.5 g sodium bicarbonate— ½ tsp.d. 2.5 ml of detergent – ½ tsp. Chill buffer in freezer or on ice for 15 minutes.2. Extraction of DNA:a. Smash banana with fork until pureed on the wax paper.b. Place a small amount of pureed banana into a clean 50 ml beaker.c. Add 10 ml of chilled buffer solutions. Stir vigorously for at least 2 minutes.d. Fit the filter paper in the funnel. Pour the banana/buffer mixture into the filter. Collect the liquid in a test tube.3. Spooling of DNA:a. Gently (tilt tube and allow alcohol to dribble down the side) add 10 ml of ice cold isopropanol to the mixture in the test tube. Since the DNA/buffer solution is denser than the alcohol, the alcohol will float on top. The boundary between the two is called an interface.b. Insert the plastic loop into the test tube. Carefully swirl the loop just below the interface. Wind (spool) the DNA that comes out of solution on to the loop.. These are not single DNA molecules, but thousands of molecules. After a minute of spooling, slowly remove the rod from the tube. The DNA, a clear, viscous, clotted mass will adhere to the rod.c. Examine and touch the DNA on the loop. Describe the DNA in your observations.4. Staining and observing:a. Add a few drops of methylene blue stain to the solution remaining in the test tube. The stain will adhere to any residual DNA that did not spool on the rod.b. You may observe the spooled DNA with a microscope by carefully applying it to a microscopeslide, adding a drop of stain, and covering with a plastic coverslip.Sources of information:Access Excellence Investigating DNA Spooling of Chromosomal DNAWhat Does DNA Look Like? genetics2Name________________________________DNA Spooling: Isolating Banana DNAStudent EvaluationObservations and Questions:1. What is the purpose of chromosomal spooling?2. What does the soap do to the cells?3. What causes the DNA to precipitate and spool on the rod?4. Describe the DNA collected during the spooling process.5. Is this a single strand of

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