Lab Competency Exam (LCE) Study Guide, Spring 2012 April 6

Knowledge Portion (~60-minutes during your regularly scheduled recitation the last week of classes)
Biology is an experimental science but you need to understand the current knowledge of the biological system you are studying and have a fundamental understanding of the techniques you are employing to properly formulate questions, design experiments and then properly analyze and interpret results. The goal of this exam is to find out if you understood what you did in the various modules this semester and why you did it. You are responsible for any background science from exercises or online lectures. You should also look at the exercise objectives. You are also responsible for knowing anything else which your lab instructor requires. Scientific Method
Given an experimental design tell me what the manipulated, response and controlled variables are and what the positive and negative assay controls would be. Given a scientific problem and a hypothesis, design an experiment that includes a description of the variables and proper controls. Know what the difference is between your hypothesis and your experimental prediction(s). When given data, be able to logically interpret it and arrive at conclusions. Be able to define each of the three types of experimental variables (i.e., manipulated, response and controlled).

Biological Molecules Module

Know the general structures and properties of the biological molecules we worked with: carbohydrates (reducing sugars and starch), proteins and fats. If given an assay name, state what specific biological molecule it tests for. If given a biological molecule, be able to tell me which biochemical test(s) would yield a positive test result. Be able to explain the fundamentals of electrophoresis (PAGE & agarose) and properly interpret gels. Know the purpose of Laemmli buffer. Know what a marker lane is and why it is used for in SDS-PAGE. If given a gel picture, be able to use the marker lane to infer protein sizes (estimate the size of a protein band by comparison to a marker lane). Know the relative migration of small versus larger proteins in an SDS-PAGE. Know how to use a standard curve to characterize an unknown. If given a gel picture, be able to determine the percent commonality between different samples. Be able to calculate percent commonality from a variety of protein profiles (i.e., raw data or results). Be able to explain how SDS-PAGE works. You should be able to explain the following: what the SDS does to the proteins; what the heating does to the proteins; why the proteins move through the gel; what the relationship is between protein size and migration through the SDS-PAGE gel; and when comparing an unknown protein band to kaleidoscope what does that tell you about the unknown. Know what type of molecule each assay (i.e., SDS-PAGE, Sudan III, Benedict, Iodine, Biuret and Bradford) will detect. Know what a positive and a negative color looks like for each assay. In addition, recall which samples would serve as negative and positive controls, for each assay.

Solutions and Serial Dilutions

Be able to apply the equations CiVi = CfVf and Vf=Vi + Vd to make different concentrations of stock solutions. Understand how to use X solutions. If given a dilution scheme and an initial concentration of a stock solution, calculate the concentrations of each dilution. If given desired concentrations of a dilution series, create a dilutions scheme to create the desired concentrations (i.e. what volumes of diluent and stocks would you use?). If given a dilution scheme and results of a bacterial enumeration experiment (dilution and spread plating of a bacterial broth culture), calculate the cell density of the original broth culture. If given the cell density of a broth culture and a dilution scheme be able to predict the results of an enumeration experiment (i.e., how many colonies would you expect to get?). If given the molecular weight of a compound, be able to make a solution of a specified molarity and volume or percentage and volume.

Spectrophotmetry
Know the nomenclature A and OD (i.e., what does the A stand for? OD stand for? Is there a difference between them? What does the stand for?). Can you explain in your own words what the Amax is for a compound? Know and be able to apply Beers Lab. When given an experiment and a sample to test, tell what solution would make a good blank for that sample and explain why.

Enzymes
Know why and how enzymes are affected by various environmental conditions (e.g. temperature, pH) and explain why they have optimum temperatures and pHs. Know why/how substrate concentration affects enzyme rates and why the rate maxs out. When given graphs, identify where the enzymes optimum environmental conditions were. When given the appropriate graph, identify when there are varying relationships between the amount of substrate and enzyme (enzyme limiting, substrate limiting or equivalent amounts).

Signal Transduction Module

Know what signal transduction means and does. Know the roles that G-proteins, ion channels and tyrosine kinases play in signal transduction. Know what second messengers are and recall specific examples. Know what a ligand is. Know what kinase cascades are and how they affect signal transduction (what is their advantage?). When given a scenario involving the use of a hemocytometer, microscope and iodine, be able to calculate the concentration of cells/ml in the original container. Be able to identify all four steps/stages in signal transduction. Also, be able to describe what is happening in each step and identify the correct order of these steps. If given a description or diagram, be able to identify all three types of plasma membrane receptors. Briefly explain how each one works when it interacts with the appropriate ligand or ligands. Lastly, be able to compare and contrast all three types with one another, in terms of: number of ligands needed, how many proteins make up the receptor, and how many pathways get turned on from each reception. Be able to define and use the following terms: relay molecules, second messengers, ligand, receptor, kinase, phosphatase, and signal amplification.

Transformation
What do we mean by a selective medium? Be able to interpret transformation results (i.e., why does the culture grow on one medium but not the other?). Be able to interpret predicted transformation results (i.e., on which media will the culture grow & on which will it NOT grow?). When given data or observations, be able to calculate transformation efficiency. What do we mean by lateral transfer of genes? Which lateral transfer method did we used in lab? What are the various methods of lateral transfer of genes that occur in nature and what are their mechanisms? Know the steps of transformation and know what is happening in the transformation tube at each step of the procedure (e.g. incubation step - what is happening when you incubate at 37oC in LB broth?) Explain how bacteria can transfer and acquire genes laterally from one cell to another. Compare and contrast plasmid DNA to bacterial chromosomal DNA.

Polymerase Chain Reaction

Know what PCR does and its applications. Know the names of the three steps of a PCR cycle. Explain what is happening to the DNA and other cocktail ingredients at each step of a PCR cycle. Know why Taq polymerase is used rather than a normal DNA polymerase. Be able to list what the ingredients are in a PCR master cocktail and explain why each ingredient is used for during PCR (think back to our experience with enzymes and what happens during in vivo DNA replication). Know what each ingredient of the cocktail is contributing to the in vitro reaction (especially the primers). To better understand the nature of the DNA molecule and its replication by carrying out its replication in the laboratory. Bring together the concepts you learned in the enzyme exercise to explain how enzymes and substrates are working to increase the amount of the PCR product.

DNA Fingerprinting & Agarose Gel Electrophoresis

If given a DNA restriction map for linear or circular DNA and a set of enzymes with which to cut the DNA, determine how many fragments are produced, what is size of each fragment produced and show where each fragment would be in a gel compared to the standard DNA (marker). Be able to compare DNA restriction maps and a gel picture of a digestion to tell me which plasmid was loaded on the gel. Know which pole DNA moves toward and why. Recall what are the three reasons for adding loading dye to your samples before loading the gel. To understand the principles behind electrophoresis, why molecules move in an electric current and how we exploit that in the laboratory. Explain why/how the ethidium bromide is used to visualize DNA fragments in the agarose. If given a gel picture of DNA samples with a marker lane, estimate the size (in base pairs) of the DNA fragments in the samples and also identify the physical form of the DNA (i.e., linear or supercoiled or nicked relaxed). To understand and explain core DNA analysis techniques: PCR, RFLP and gel electrophoresis Compare and contrast restriction endonucleases and Taq polymerase, in terms of: what each one does, what type of DNA each one acts on, and what environmental conditions each functions in. Be able to list what are the ingredients used in a restriction digest and explain why each ingredient is present (what is its function). To be able to explain and apply cellular processes that involve DNA: DNA replication and the bacterial restriction-modification system. When given DNA data, bioinformatics and other information, identify the unknown organism. Compare and contrast agarose gel electrophoresis and SDS-PAGE, in terms of: gel component, biological molecule separated, direction of the gel, movement of the electrical current, marker used and relationship between molecule size and distance migrated. Compare and contrast protein and DNA fingerprinting. Bring together the concepts you learned in the enzyme exercise to explain how enzymes and substrates are working to create different sized fragments of linear DNA. When given any of the media we used for biochemical testing, be able to explain why an organism would grow on each type of medium and why it would have a specific type of colony coloration or cause a change in the medium color.

Practical - Bench Skills Portion (~50 min completed during your scheduled lab the last week of classes)
Biology is an experimental science and proficiency at fundamental bench skills is critical for data to be reliable and valid. Proficiency at these skills will also benefit you in future lab courses and in your science careers. We will test this with a laboratory practical exam. A series of stations will be set up asking you to properly perform some basic bench skills. You will be given a fixed amount of time at each station (4 minutes) before moving on to the next station. Nine Stations (not necessarily in this order) 1. Plating Techniques (inoculating agar plates by streak and spread method) - 4 min 2. Dilutions and Solutions 4 min 3. Gel electrophoresis = setting up the box and power supply (agarose and SDS-PAGE) - 4 min 4. Gel electrophoresis = loading (agarose and/or SDS-PAGE) 4 min 5. Graphing - 8 min 6. Micropipetting - 4 min 7. Microscopy - 4 min 8. One station to perform calculations to prepare solutions and/or make dilutions - 4 min 9. Serological pipetting - 4 min Techniques to master Be able to use micropipettors properly to accurately measure a variety of volumes of liquid to a new container. Be able to use serological pipettes properly to accurately measure a variety of volumes of liquid to a new container. Be able to identify which micropipettor or serological pipette should be used to measure a specific volume of liquid accurately (recall the ranges each measuring tool should be used to accurately measure volumes). Be able to transfer liquids and perform dilutions to create new solutions or to transfer volumes of liquid. Be able to calculate and prepare a solution using the equations: CiVi = CfVf and Vf = Vd + Vi Be able to convert between microliters and milliliters. Be able to properly place the comb and connect the electrodes properly on an agarose gel and an SDS-PAGE. If given an electrophoresis set up, agarose or SDS-PAGE, be able to state whether it is set up properly so it will work or not and if not, then explain what is wrong and why it will not work. Be able to successfully load ONE well in an agarose gel and/or SDS-PAGE, and determine which well each sample should be placed in for it to migrate in the correct direction in the apparatus. Be able to list the differences and similarities between an agarose gel box and SDS-PAGE box. Be able to focus a microscope, to do the following: draw the organism, measure the size of the organism, make a wet mount to determine if organisms are moving or not. Be able to calculate total magnification on the compound microscope. Be able to identify different parts of the microscope and explain how to use each to focus on a specimen (i.e., objective lens, ocular lens, condenser, iris diaphragm, coarse adjustment knob, fine adjustment knob, stage). Be able to streak an agar plate to obtain isolated (i.e. pure culture) colonies without contaminating the work area. Be able to properly perform the spread plate technique to get a lawn of growth without contaminating the work area. Be able to properly perform the aseptic technique before performing a plating technique by selecting and/or identifying which tools and materials are used for each technique. When given plates with bacterial growth, be able to identify the different patterns of bacterial growth (e.g., lawn versus isolated colonies). Be able to determine which plate should be used when wanting to use a selective or non-selective or differential or a combined medium. Also, identify if bacterial transformants have grown or not on different types of media. Other bench skills or practical knowledge which your lab instructor ask you to know. Graphing Skills and Abilities Be able to use MS-Excel (97 version or older) to make and label a graph from a given set of data. Know what a standard curve is and what it is used for (remember, not all graphs are standard curves). Be able to create time course curves and optimal curves (be sure to insert the appropriate line). When given a data table, be able to use MS-Excel to graph the data points. Be able to able the axis properly. Be able to set the scale of an axis (linear or logarithmic). Be able to set grid lines properly (both horizontal and vertical). Be able to the graph a proper title. Be able to add the most appropriate trendline or best-fit line. Be able to display the equation for that line. Recall semi-log versus regular graph scales (be able to pick the most appropriate scale for the data provided). Be able to use the graph to identify information about unknowns.