BIO304_labNotebook_week1_LabOlympiad

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Table of Contents Week 1: Lab Olympiad! 1 Week 2: The Fruit Fly Identification Crisis (Lab 1) 10 Introduction: 10 Before you begin: 10 Become familiar with the equipment on your bench 10 Materials provided to you for this lab: 10 Procedure: 10 Think about it! 12 Remember Mendel's Peas? 12 Apply the ideas of transmission genetics to the "Fruit Fly Identify Crisis" 14

Week 1: Lab Olympiad Lab Safety Today, you will be working with strong acids and bases. It is important to protect your skin by wearing shirts that cover all of the abdomen, long pants, closed-toed shoes, and gloves when handling these chemicals. If you do not have the proper clothing on, please go change! Learning Objectives: 1. Become familiar with the safety procedures and equipment for the laboratory. 2. Review the metric system and how to make unit conversions. 3. Learn how to choose and use the appropriate pipettes. 4. Apply what you have learned by performing a serial dilution. Introduction To maximize your success and safety in the lab, you must know where to find the things you need, how to use them properly, and how to keep yourself and others safe. Today's activities will guide you through critical knowledge that will set you up for success for the rest of the semester. Since these tasks may seem mundane, we're making this a competition! You must complete each task AND have your TA approve its completion before moving on to the next task. The first table that has completed all tasks wins! Rules of the Lab Olympiad 1. Perform all events in order. 2. Have your instructor sign off on each event BEFORE moving onto the next event! 3. If your group fails any part of an event, you will need to go back and repeat the part that was not done correctly. 4. Have fun and be safe! Event 1: Lab Safety and Rules Working in a laboratory can be exciting and fun while also help you learn the content from class. But all labs have certain hazards that you need to pay attention to. All labs have rules and equipment for handling potential hazards and managing dangerous situations. Each week, the lab will include specific safety instructions that includes specific protective gear such as gloves as well as how to dispose of any waste we create during the lab. There are also general safety rules that you should follow whenever you come to lab that are listed below. General Safety Rules 1. Always wear closed-toed shoes, long pants, and shirts that cover the entire abdomen (you don't want acid to burn the skin on your stomach!). If you have long hair, it is best to tie it back to keep it from coming into contact with chemicals or equipment. 2. NO food or drinks in the lab! All food and drinks must be kept in closed containers in your backpack/bag OR can be kept in the hallway. 3. Learn the location of all safety equipment such as the fire extinguisher, eyewash station, and first aid kit. 2

4. Ask for clarification or help if you don't know how to handle something, that's what your instructor is here for! 5. Be alert. Be watchful for spills or leaks. Listen for sounds that are not normal, such as malfunctioning equipment. 6. Clean up your work area as needed during and after the lab. 7. Dispose of used chemicals and supplies in the designated waste containers. 8. Wash your hands before you leave. Proper Disposal This laboratory is equipped with a variety of equipment and signage with emergency instructions as well as containers to dispose of reagents appropriately on the benchtop and around the room. Here are some common things you will be exposed to:  Pipette tips and small tubes can typically be disposed of on the benchtop as can many (BUT NOT ALL) of the liquid reagents that you will use in this lab. At the end of the lab, benchtop waste containers should be emptied into the regular trash.  Bacterial and yeast cultures should always be disposed of into the biohazard waste containers. Safety Items and Signs Find the following Safety Items/Signs and describe where they are. q Chemical fume hood: q Fire extinguisher q Eye Wash q First Aid Kit q Signs with Emergency Instructions q Biohazard Waste Containers q Regular Trash Receptacles Lab Safety Situations Read each of the following situations and come up with a resolution. When you have resolved all situations, ask your TA to check your answers. Situation 1: I just got a coffee on my way to lab! I don't want to waste it, but I know that I can't bring it into the lab. What should I do!?! Situation 2: I hear a loud high pitched sound coming from the centrifuge. What should I do? Situation 3: It is hot out today and I have shorts and a tank top on. We are only working with fruit flies today. Do I need to change? 3

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Situation 4: The lab protocol doesn’t give instructions on how to dispose of some of the chemicals I'm working with today. Can I just pour them down the drain? Situation 5: I just realized that I touched near my eye with my gloves on! We're working with weird bacteria today. What should I do? Situation 6: The group behind me is being reckless and one of them just got a cut on their finger. What should I do? Ask your TA to signoff before moving on to the next step! Event 1 TA signoff: _____________ Event 2: Review the Metric System Scientists worldwide typically rely on the metric for measurements of length (meters), mass (grams), and volume (liters). They also measure temperature in degrees Celsius. Working as a team, fill in the blacks below. Write the numerical and equivalents for each! You can use the internet to convert Celsius to Fahrenheit, but don't use When you have completed them all, ask your TA to check your work. 1. 5mL = ___________ L = ___________ uL 2. 500uL = __________mL = ___________ nL 3. 0.25mL = _________ uL = ___________ L 4. 25uL = ___________mL = ___________ nL 5. 1.5mL = __________uL = ___________ L 6. 25 C = _________ F 7. 37 C = _________ F 4 Unit Symbol Relationship Liter L Milliliter (10 -3 L) mL 1000mL = 1L Microliter (10 -6 L) uL 1000uL = 1mL Nanoliter nL 1000nL = 1uL

Ask your TA to signoff before moving on to the next step! Event 2 TA signoff: _____________ Event 3: How to choose and use pipetting devices to measure liquids Types of Pipets and Pipetting Devices Liquid volumes can be measured in various ways. In this lab, we primarily need very small amounts of liquids to complete the experiments that are below 1ml (1,000 uL or less). Therefore, you will typically be using micropipettes and pipette tips. Pipettors can be set to a very specific volume within a given range. It is important to stay within the range of the pipettor as going outside of the range will change the calibration of the pipettor making future measurements inaccurate. As a result, experimental results are unreliable and some of the labs might not work at all! The pipettors you will be using in this laboratory have the following ranges: P20 (2-20uL), P200 (20- 200uL), P1000 (100uL-1000uL). When choosing a micropipette, you need to consider both the range of the instrument and its ability to measure the amount accurately. Suppose you want to measure 125uL. Both the P200 and P2000 can measure 125uL. However, the P200 would have a smaller percent error because it can read to the nearest 1uL while the P1000 can only be read to the nearest 10uL. Therefore, it would be better to use the P200, especially if accuracy is important. To properly measure liquid using micropipettes , you need to choose the correct pipettor for the measurement amounts, the correct tips, and use the micropipettes properly. The tips for the P20s and P200s are smaller (and yellow). The tips for the P1000 are larger (and blue). To measure liquid, you will need to complete the following steps: 1. Choose the correct micropipette for your volume and set to the correct number. 2. Attach the pipette tip by placing the end of the micropipettes into the tip and tap gently. Then lift out of the box. 3. Push the button on the top of the micropipette to the 1 st stop ("soft stop" in picture below) while the tip is in the air. 4. Place end of tip into the liquid and slowly release the button to draw up liquid. 5. Move to the receiving container, place tip against the wall of the receiving container. 6. Push the button on the top of the micropipette all of the way down ("hard stop") to ejec t the liquid. Keep the button pushed down while you pull the micropipette and tip out of the receiving container. 7. Eject the tip into the benchtop waste container. 5

Lab Event 3 Questions and Activities: Discuss the following questions with your team and record your answers below. 1. Sam wants to measure exactly 205.5uL of water and gets a P200 for the task. Is his choice of micropipette suitable for this amount? Why or why not? Explain your reasoning. 2. Bonnie wants to prepare a mixture of liquids A-E. She needs the amounts specified below. For each liquid, determine which micropipette would be best to use. Liquid mL Needed Conversion to uL Measuring Device to Use and Why A 0.4 B 0.015 C 0.070 D 0.005 E 0.025 3. Consider the the following images of the volume dials of a P1000 and P20. For each, what volume of liquid is specified (in uL)? ____uL ____uL ____uL ____uL ____uL ____uL ____uL ____uL ____uL 4. Inspect a P200. Can a volume of 98uL be accurately measured with this device? 5. Fill out the image to the right to ensure that you are familiar and comfortable with working with the micropipettes. Note that not all of these parts have been described above. Explore the pipettes in front of you and determine what you think each part should be called. (While you explore the pipette, remember to treat them with care! They are expensive and easy to break!) 6

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Ask your TA to signoff before moving on to the next step! Event 3 TA signoff: _____________ Lab Event 4: Liquid Measurements with Micropipettes Supplies needed for this event:  Large test tube rack  7 large test tubes  40 mL of "Cabbage Tea"  Tube of vinegar (acid)  Tube of NaOH (base)  Waste container for used tips  Microcentrifuge test tube rack  Microcentrifuge tubes  Tube of blue dye  Tube of water Part 4A: Color Changing Cabbage Tea! Working as a team, perform the following measurements. Subdivide the work so that everyone uses the micropipettes. Everyone on your team should become comfortable and skilled in using the micropipettes since we will be using them throughout the semester. 1. Place 7 large test tubes in the test tube rack. 2. Add 5mL of the blue cabbage tea to each tube. The cabbage tea is made using red cabbage and can be used as a pH indicator (how cool is that!) 3. Following the table below, add the specific amounts of acid or base to each tube. Determine the amount of water to add to keep the final amount consistent across all tubes. Tube # 1 2 3 4 5 6 7 Acid 0.820mL 0.063mL 0.007mL none none none none Base none none none none 0.007mL 0.063mL 0.820mL Amount of water to add Color at the End 4. Gently twirl the tubes to obtain a consistent color throughout each tube. Then fill out the color you observe. Part 4B: Small measurements The smallest volume that you pipetted in 4A was 630uL. Many experimental protocols you will complete in this lab require measuring even smaller volumes. Each member of your group will complete the following. Note: The dye contains bromophenol blue , which will stain your skin and clothes. 7

1. Label 5 microcentrifuge tubes 1-5. 2. Add the amounts of dye and calculate the correct amount of water to add to make each volume equal to 200uL. Tube 1 2 3 4 5 Dye 40uL 20uL 10uL 5uL 2uL Water 3. Compare the color in your tubes. Do you observe the same total volume of solution in each tube? Is the color intensity in each tube of the series the same for all members of your group? If you see obvious differences, why could they have happened? 4. If you are satisfied with what you observe, ask your TA to come over to inspect them. Note that your TA may have you repeat the activity if they see that something did not go according to plan! Ask your TA to signoff before moving on to the next step! Event 4 TA signoff: _____________ Lab Event 5: Serial Dilutions A serial dilution is a standard laboratory technique used to obtain a desired concentration less than that of the original solution or starting sample. Dilutions are always ratios and are generally expressed in terms of whole numbers (reduced to the lowest common denominator). The dilution ratio is defined as the volume of sample per total volume (total volume = sample + dilution solvent). The image to the right is an example of a 1/10 serial dilution of a bacterial sample. Note that when the bacteria are grown on a plate, the number of colonies decreases approximately 10x with each dilution. The image below shows the dilution being made with 1mL of sample and 9mL of new broth for each dilution. A 1/10 dilution could also be made using other measurements such as 2mL sample and 18mL broth. Both would be examples of a 1/10 dilution and would have the same concentrations, the only difference is the total volume made with each dilution. Note that the image above shows a 1/10 dilution, but serial dilutions can be made in other ways. Some dilutions could be less extreme (e.g. 1/2 dilution or 1/4 dilution) while others can be more extreme (e.g. 1/100 or 1/1000). Lab Event 5: Serial Dilutions Part 5A: Make drawings of serial dilutions 1. Suppose you have 10mL of a concentrated solution of dye and want to prepare a 1/3, 1/9, and a 1/27 dilution so that you have at least 5mL (you can have extra!) of each solution to work with when you are all done. How 8

can you accomplish this using a serial dilution? Draw a scheme that illustrates your method (include shading to illustrate how concentrated the solution is in each tube). 2. Draw a scheme that illustrates using 100uL of dye and 100uL of water to create a serial dilution with 3 dilutions. What is the concentration of each tube compared to the original concentration? What is the final volume of each tube that you produce? Draw a scheme that illustrates your method (include shading to illustrate how concentrated the solution is in each tube). Ask your TA to signoff before moving on to the next step! Event 5A TA signoff: _____________ Part 5B: Make serial dilutions Each group member should complete the serial dilutions you drew out for 4A number 2 using the microcentrifuge tubes on your benchtop, the dye you used before (concentrated tube), and water from before as well. Remember: Loading buffer contains bromophenol blue , which will stain your skin and clothes. Once you have completed the serial dilutions, compare them to those made by your other group members. Compare the color in your tubes. Do you observe the same total volume of solution in each tube (should you?)? Is the color intensity in each tube of the series the same for all members of your group? If you see obvious differences, why could they have happened? If you are satisfied with what you observe, ask your TA to come over to inspect them. Note that your TA may have you repeat the activity if they see that something did not go according to plan! Ask your TA to signoff before moving on to the next step! Event 5B TA signoff: _____________ Lab Event 6: Clean up your space! q A critical part of working in a lab is leaving the space clean and safe for others to be able to use it. To clean up from this lab, complete the following checklist: q Add all liquid waste from the acids and bases experiments to the liquid waste beaker on your benchtop, then pour down the drain. q Rinse out the tubes and leave upside down to dry for the next group to be able to use. q Throw away all used microcentrifuge tubes and tips in the regular trash as well as any used gloves. q Use the ethanol in the spray bottles and a paper towel to clean your benchtop space. q Check the area to make sure there is nothing else to tidy before you leave. 9

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Ask your TA to signoff before leaving the lab! Event 6TA signoff: _____________ Congratulations! You have completed the Lab Olympiad and your first BIO 304 lab! 10

Week 2: The Fruit Fly Identification Crisis (Lab 1) Introduction: Someone has lost all the records for our laboratory lines of Drosophila melanogaster and the vials containing these flies have lost their labels!! Your assignment is to help us begin to recover the missing information and identify what kind of flies are in each vial. You have been given a vial that contains BOTH the “ wild type ” line and a vial with flies containing an unknown fly line. Before you begin: Become familiar with the equipment on your bench Your bench contains a dissecting scope, a paint brush, a fly pad, and a wand connected to a CO2 tank. The wand is used to anesthetize flies in vials. The pad is used to keep flies anesthetized while you observe them. The paint brush is used to move flies after they are on the pad. The dissecting scope you will use in this lab has two settings for magnification, 1x and 3x, for making observations. You should first focus the microscope using the focus knob to move the scope up and down while on the 1x magnification. Different people prefer different magnifications, for initial sorting of the flies by larger phenotypes (e.g. body color), you might prefer the 1x magnification. When you need to "take a closer look", you will want to use the 3x magnification. If the microscope was in focus, but looks blurry or weird when you change the magnification, check that you are not "between" magnifications by turning the magnification knob at the top of the scope. Note that the light source above the stage of the microscope is controlled by the knob on the base on the right. There is also second light source on the stage is controlled by a switch at the right side of the base. Materials provided to you for this lab:  Dissecting microscope  CO2 tanks, wands, and pads  1 vial containing a 2 different Drosophila lines Procedure: 1. Anesthetize the flies in one of the vials containing 2 lines of Drosophila fruit flies a) The flies are anesthetized but not killed by CO2. The CO2 tank at your bench is not to be adjusted by you. The only manipulation of CO2 flow that you may need to perform is to shut off the flow of CO2 to your wand after you have dumped anesthetized flies on your fly pad. This is accomplished by turning the white-marked knob on the connector at the base of the tubing connected to your wand. b) Make sure CO2 is flowing from the wand and the fly pad. Turn on the light on the dissecting scope. c) Gently tap the vial on the table to knock the flies away from the plug, Hold the vial at the bottom, near the food, in a horizontal orientation. Use a finger and the CO2 wand to push the plug away from the side of the vial so the wand can be inserted into the vial. It should only take a few seconds to knock the flies out. YOUR 11

GOAL IS FOR THE FLIES TO FALL TO THE SIDE OF THE VIAL WHEN THEY ARE KNOCKED OUT. Anesthetized flies that get into the food will likely get stuck. d) Keep the vial horizontal and twist and pull out the plug. Dump the flies on the fly pad and observe them in the dissecting scope at it lower magnification setting. If flies are standing up or walking around, you should cut off the CO2 flow to your wand to increase the CO2 flow to the pad. Ask the laboratory instructor for help if needed. 2. Observe the flies! The paint brush can be used to sort or reposition anesthetized flies on the fly pad. Use the brush to sweep or push flies but DO NOT CRUSH THEM OR IMPALE THEM ON THE BRUSH BRISTLES. You should keep the flies alive during observation. 3. Drosophila males and females are sexually dimorphic (the sexes look slightly different). One obvious difference is that the males are smaller than the females. There are also two different lines of Drosophila in your vial. These lines have 3 trait differences that you can observe in these flies. What trait differences do you see? List them here. 4. Make 6 piles of flies based on trait differences and what you think could be males vs females. All of the flies in each pile should look almost identical! When you are satisfied with your piles (including separating males and females into different piles), ask your TA to check your work. Ask your TA to signoff before moving on to the next step! TA signoff: _____________ 5. While both fly lines are on your pad, compare the phenotypes of wildtype (WT) and your Unknown flies. Assume that one gene controls each trait and write a hypothetical genotype for the WT and the Unknown strains for each phentoype. Designate a gene name and symbol (a letter or two or three) for the gene using the following notes and rules: All wildtype flies have red eyes. Name the gene that controls the trait after the form of the trait in the Unknown. In Drosophila genetics, the wildtype genotype includes a "+" allele For example, if the WT has two wings and the Unknown has six, then the gene could be called “six wings”, and the gene symbol could be “sw”. The WT genotype would be sw+ sw+ and the Unknown genotype would be sw sw (this example is not a trait that you see in your flies) Observations Gene Name Wild type genotype Unknown genotype Example 2 wings vs 6 wings Six wings sw+, sw+ sw, sw Trait 1 Trait 2 Trait 3 12

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Ask your TA to signoff before moving on to the next step! TA signoff: _____________ Think about it! The "Fruit Fly Identification Crisis" is a genetic analysis of morphological phenotypes using the fruit fly, Drosophila melanogaster . Together with your group, you will apply the principles of transmission genetics to solve this problem. That sounds intimidating! BUT most (if not all) of you have done this work before in BIO 148 😀 Complete the following problem sets to remember what you already know. First think about transmission genetics using Mendel's peas as an example. answer the questions regarding transmission genetics as they pertain to the "Fruit Fly Identification Crisis" Remember Mendel's Peas? Gregor Mendel was a monk who lived in the 1800s. He was curious why pea plants sometimes produced plants and peas with different traits. He designed simple but effective procedures to study the patterns of inheritance for heritable traits using pea plants. We still use these procedures today (including throughout our "Fruit Fly Identity Crisis" lab series). Remember that Mendel's procedure included using pure-breeding lines of peas that different from each other in different phenotypic traits. If the phenotypes are always identical, what does that mean about the genetics of those lines? (Think about if dominance matters and/or if being heterozygous/homozygous matters) Now I want you to think about Mendel's Law of Segregation . Remember that the Law of Segregation requires only 1 gene in order to observe it. Let's use seed shape as the gene that we will use to explore this idea. Since the dominant phenotype is round, let's use the symbol R for the round allele and r for the wrinkled allele. Answer the following questions to track the inheritance of the R and r alleles. Draw out the cross when you breed the 2 pure-breeding lines. Genotype and Phenotype of: Round Parent ________ X Wrinkled Parent __________ The F1 offspring of a pure-breeding cross will result in genetically identical offspring. What is the phenotype and genotype of the F1 offspring? 13

What happens when you cross 2 F1 offspring to each other? Show your work below. Include a Punnett Square, the ratios of different genotypes AND the ratios of different phenotypes. Now I want you to think about Mendel's Law of Independent Assortment . Remember that the Law of Independent Assortment requires at least 2 genes in order to observe it. Let's use seed shape AND seed color as the genes that we will use to explore this idea. Draw out the cross when you breed the 2 pure-breeding lines that differ in 2 genes. Genotype and Phenotype of: Parent with Round and Green Seeds __________ Parent with Wrinkled and Yellow Seeds __________ The F1 offspring of a pure-breeding cross will result in genetically identical offspring. What is the phenotype and genotype of the F1 offspring? What happens when you cross 2 F1 offspring to each other? Show your work below. Include a Punnett Square, the ratios of different genotypes AND the ratios of different phenotypes. Remember that 2 genes will result in MANY genotypes and 4 phenotypes. 14

Ask your TA to signoff before moving on to the next step! TA signoff: _____________ Apply the ideas of transmission genetics to the "Fruit Fly Identify Crisis" 1. What could you do to test if the phenotypes that you observed above are genetically based? 2. How could you determine the dominance relationships between the traits in the WT and Unknown flies? 3. Suppose that one of the wildtype traits is dominant. Does that mean that ALL wildtype traits must be dominant? How could you test this? 4. What would you do to determine if one or more of the traits you observe is sex-linked, and what is the predicted result if none of the traits are sex-linked? 15

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BIO304_labNotebook_week1_LabOlympiad

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