Exercise 1 – extraction DNA from cells
Summary of extraction method: Onion cells are difficult to lyse (rupture) because plant cells are surrounded by a cell wall (a tough barrier made of cellulose). (1) Breaking up the onion in a blender helps to release the cellular contents. (2) The chopped material is then mixed with lysing solution, which contains (a) a detergent to break open nuclear membranes, (b) sodium chloride to clump DNA molecules so they will precipitate out when alcohol is added, and (c) a citrate ion to prevent any DNAase in the cells from digesting the DNA.
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(3) The heat treatment of 65° C is just hot enough to denature the DNAase (a protein) without degrading the DNA (a nucleic acid). (4) The plant material debris is then filtered off and cold alcohol added to the filtrate. DNA is soluble in water but insoluble in alcohol, so the DNA in the filtrate precipitates out along the interface where the filtrate makes contact with the alcohol. When a spooling rod is inserted into the interface and rotated, the precipitated DNA fibers wrap around the rod. Extraction procedure (Work in pairs!) In the Central Study Area transfer 7 ml. of the liquefied onion to a 20-ml test tube. Pouring onion sludge into the small opening of the tube is tricky, so use a small beaker to fill the test tube about one- third full. Add 7 ml cell lysis solution to the tube. When this is added, the 20-mL tube should be two-thirds full. Hold your thumb over the top and invert the tube once or twice to mix the contents. Do not shake it. Place the tube in a water bath at 65° C for 15 minutes. (While waiting, practice pipetting by doing Exercise 2.) Remove the tube from the water bath. Place a funnel into the mouth of a clean, larger (35-ml) test tube. Use a small piece of cheesecloth to act as a filter in the funnel. Slowly pour the onion solution into the funnel, letting it drip for a while. If necessary, squeeze the liquid through, but do not allow solids to fall into the test tube. Obtain 10 ml. of the chilled DNA precipitation solution (95% ethanol). Hold the tube containing the lysate (the solution of onion DNA) at a 45° angle and slowly allow the precipitation solution to slide down the surface of the tube to form a nice layer on top of the lysate. Observe the DNA as a cloud of fine, viscous threads where the 2 liquids meet. This is called the interface. Insert a glass rod into the test tube until its tip is just below the interface. Turn and twist the rod, gently passing it back and forth between the 2 layers of liquid. The DNA should wrap around the rod like yarn on a spool. List several adjectives to describe the DNA you have extracted (show it to your TA): DO NOT USE the extracted onion DNA for the DNA fingerprints in Exercese 3. For that exercise you will be given fresh samples of “crime scene” and “suspect” DNA.