Bio5LAManual12f

5m stock that will be needed to make the required

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Unformatted text preview: s complete, the two groups should share the data for the solute that they did not use. 1. Obtain 7 – 18x150 size test tubes and label each with one of the concentrations listed in the second column of Table 3. Biology 05LA – Fall Quarter 2012 Lab 3 – page 7 2. Keeping in mind that you will be given a 0.5 M stock solution (C1) and that you will be making 10.0 ml (V2), of each concentration, you should now use C1V1 = C2V2 to solve for V1 (the volume of the 0.5M stock that will be needed to make the required dilution). Once calculated, record these values for V1 in the correct cells of Table 3. Desired TONICITY molarity (C2) Hypotonic (V2 - V1) 0.0 0.15 4. Make the required dilutions. Your TA will instruct you on how to do this efficiently. 6. Close each tube with Parafilm, and mix by gentle inversion. Allow the samples to stand 5 minutes before collecting data. V1 (ml) 0.1 3. Make the simple calculations needed to fill in column 4 of the table. 5. Add 0.4 ml of the RBC suspension into each of the tubes (remember to re-suspend the cells before dispensing). ml H2O to be added 0.20 0.25 Hypertonic 0.30 0.35 Table 3. Dilution Work Sheet Data collection. What you need to find here is the lowest solute concentration that contains intact cells. In order to do this you will need to go back to the tubes made in Part 1 of this exercise and ask two questions: which of these tubes contain intact cells and is there any apparent difference between the tubes with intact cells verses those with lysed cells? You should see that there is an easy way to tell the difference. Analysis. Using the visual feature alluded to above, what was the lowest glucose concentration at which intact cells were present? What was the lowest NaCl concentration at which intact cells were present? Explain this difference (remember that the values used are approximations). Learning Goals/Desired Outcomes. 1) Be able to define and differentiate between the following pairs or groups of terms: a) solute - solvent. b) diffusion - osmosis. c) hypotonic - isotonic - hypertonic. d) molarity - osmolarity. 2) Be able to define the following terms: a) concentration gradient. b) differentially permeable membrane. c) tonicity. 3) Be able to explain how variations in solute molecular mass, solute concentration, and temperature influence diffusion rate. 4) For an aqueous system where two solutions are separated by a differentially permeable membrane, you should be able to predict the direction of osmosis given the molar and/or osmolar solute concentrations on both sides of the membrane. 5) You should be able to explain why osmolarity differences are more significant than molarity differences when trying to predict the direction of osmosis. 6) You should be able to predict how plant cells will respond to exposure to a hypertonic environment and to explain the reasoning behind your prediction. 7) You should be able to predict how animal cells will respond to exposure to hyper- and hypotonic conditions and to explain the reasoning behind your predictions. 8) For the exercise where you estimated the osmolarity of some red blood cells, you should be able to explain why the lowest solute concentration at which the cells were unbroken represented a reasonable estimate of the osmolarity of the cells. Bio 05LA – Fall Quarter 2012 Lab 8 This portion of the Genetic Analysis lab will be performed along with lab 4. Lab 8: Genetic Analysis Laboratory - Detection of Alu Sequences by PCR (Part I) To obtain DNA for use in the polymerase chain reaction (PCR) you will extract the DNA from your own living cells. It is interesting to note that DNA can be also extracted from mummies and fossilized dinosaur bones. In this lab activity, you will isolate DNA from epithelial cells that line the inside of your cheek. To do this, you will rinse your mouth with a saline (salt) solution, and collect the cells using a centrifuge. You will then boil the cells to rupture them and release the DNA they contain. To obtain pure DNA for PCR, you will use the following procedure: The cheek cells are transferred to a microcentrifuge tube containing InstaGene™ matrix. This particulate matrix is made up of negatively charged, microscopic beads that chelate, or grab, metal ions out of solution. It traps metal ions, such as Mg2+which are required as catalysts or cofactors in enzymatic reactions. Your cheek cells will then be lysed, or ruptured, by heating to release all of their cellular constituents, including enzymes that were once contained in the cheek-cell lysosomes. Lysosomes are sacs in the cytoplasm that contain powerful enzymes, such as DNases, which are used by cells to digest the DNA of invading viruses. When you rupture the cells, these DNases can digest the released DNA. However, when the cells are lysed in the presence of the chelating beads, the cofactors are adsorbed and are not available to the enzymes. This virtually blocks enzymatic degradation of the extracted DNA so you can use it as the template in your PCR reaction. You will first...
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