SBI4U: Microscopic Titration and Extraction of Chromatin from Liver

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Meghan Blake
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1 SBI4U: Microscopic Titration and Extraction of Chromatin from Liver Assigned: Due: Received:

2 SBI4U Microscopic Titration & Extraction of Chromatin from Liver Background We have 46 chromosomes in the nucleus of all of our cells (except reproductive ones). These chromosomes are usually in the form of chromatin which is made of strands of DNA wound around histones creating a beads on a string motif. Extraction and purification of DNA is critical to research, paternity testing and to criminal investigations. Once extracted, we can use the DNA to make genetically engineered medicines, find out who the real father is (Maury would be lost without this!), or identify the real perp or even set the wrongly- convicted free! Purpose The purpose of this investigation is to come to understand and explain how DNA is extracted by combining all of our knowledge of biochemistry with actual experience. More precisely, we are investigating and explaining why certain chemicals are used in each stage of the extraction of DNA. Furthermore, the step- by- step titration (gradual addition of detergent) and investigation under the microscope will enhance your actual understanding of what is actually happening to the cells and the chromatin as you go through the procedure. Procedure 1. Place a small piece of liver (about 1 cm X 2 cm) into a mortar and pestle. 2. Cut the liver into pieces as small as possible. 3. Add 50 ml of 0.9% NaCl solution to the well diced liver. 4. Grind the tissue thoroughly for about 5 minutes using a circular motion. 5. Strain this mixture through J- cloth or cheesecloth in a small beaker. 6. The liquid that comes through is called the cell suspension and should have a chocolate- milk like appearance. 7. Using a medicine dropper, remove a small drop of the cell suspension, place on a slide and add a small drop of methylene blue. Add a cover slip and observe the cells under low power. And then medium power. Give a general description of the mixture and provide a sketch of the appearance of the cells under the microscope in your results table. 8. Add 2 ml of detergent solution to the liver suspension. Gently mix this and then repeat step 7. If you are lucky, you might catch the detergent punching holes in the membrane and see the cytoplasm spilling out. Watch for this! 9. Repeat step 8 until very few intact cells are visible. This solution is called a cell lysate. 10. Gently pour about 50 ml of 95% ethanol into the test tube. Spool some chromatin onto a glass rod or wooden stir stick and repeat step 7. Results Create a table with the following headings: Number of Detergent Additions General Description of Mixture in the beaker Sketch of Appearance of what you see under the microscope Be ready to add as many rows as necessary until the cells are completely lysed.

3 ANALYSIS 1. Examine the two segments of double stranded DNA in the image below. If these were dissolved in pure, distilled water, and floated near each other, would they attract or repel each other? Concisely but accurately, explain your choice.

4 Why suspend the liver cells in salt solution? Why not just use water? Examine the segment of DNA in the image below. Consider that after complete lysis, the DNA is exposed to the dissolved sodium ions and chloride ions in addition to the water molecules in the 0.9% NaCl solution. What is the total, overall electric charge on the segment of DNA in the diagram below? (PRIOR TO SEEING NaCl) DNA segment Sodium ions Chloride ions Water molecules Would sodium ions or chloride ions form an ionic attraction with the phosphate groups of each base pair? How many of these ions be required to neutralize the overall charge on this segment of DNA? Draw the correct ion near the part of the phosphate they would be most attracted to in the diagram to show how this would happen. What is the name of the attractive force between the salt particle(s) and the phosphates? What kind of intermolecular force would exist between the water molecules the DNA is floating in and these salt ion/phosphate complexes?

5 Freeing the Chromatin! Do some review and research into animal cell structure. How many total membranes need to be lysed in order to free the chromatin from the cell? HINT: do some precise research into the structure of the nucleus to help guide you to the correct answer. The detergent molecule was a substance called sodium dodecyl sulfate or SDS or sodium lauryl sulfate (SLS). Do some research and show the structural formula of the detergent molecule. Cite your source using APA formatting. Do some research and briefly describe in your own words, how detergents like SDS cause cells to lyse. Cite your source using APA formatting.

Precipitating the DNA Why does ethanol cause this to happen? Redraw in the salt ion/phosphate complexes on both strands but keep them small, you ll need room for the ethanol molecules.

6 Precipitating the DNA Why does ethanol cause this to happen? Redraw in the salt ion/phosphate complexes on both strands but keep them small, you ll need room for the ethanol molecules. The chemical structure of ethanol is shown below, along with a ball and stick and a condensed version of the molecule. Chemical structure Ball & Stick Condensed structure For the chemical structure, circle the polar parts of the ethanol molecule in blue and the non- polar parts of the ethanol molecule in red. What intermolecular force would be the strongest possible between the salt/phosphate complexes and the ethanol molecules? Draw in the ethanol molecules into the diagram above, showing how they coat the DNA. Use either of the three styles from the table above. Draw the ethanol molecules oriented to maximize this attraction.

7 So, why would adding ice- cold ethanol to the cell lysate cause the DNA to clump together and precipitate? (i.e. no longer be soluble in water) CRITERIA LEVEL 4 LEVEL 3 LEVEL 2 LEVEL 1 < LEVEL 1 Thinking & Investigation - application of skills and (analysis, reasoning, interpretation, problem- solving) - applies all or almost all of the skills and with a high degree of accuracy and precision - applies most of the skills and with an adequate accuracy - applies some of the skills and with a moderate accuracy - applies few of the skills and with a limited accuracy - applies very little skills and with a minimal accuracy Communication - accuracy and effectiveness of scientific terminology, symbols, and conventions - clarity of expression shows a high shows a considerable shows a moderate shows a limited shows a negligible Staple your results table and APA formatted reference list to the back of this package.

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