Genetics lab C.elegan worms

Genetics lab C.elegan worms

The lab in question is withing the file, use interpreted data to complete the lab report and be detailed in the math and thought process involved. The Grading rubric is also included

1. elegans Project Lab Report

 

Report Components (See rubric for more details):

 

Introduction: Provide background information on the project. Remember to include information on the specific mutation you investigated. Clearly state your scientific question and your hypothesis.

Include the following: A detailed paragraph of C. elegans, their life cycle, where they are naturally found, what they eat, ect. A paragraph on distinguishing between males and hermaphrodites (adult and L4). Be sure to mention the use of the Him-8 strain, and its purpose for this experiment in the sex paragraph. A paragraph about your mutant worm-what gene was mutated (include gene name and allele), what protein (if known) does it encode, what happens to cause the phenotype you saw, do some research on this gene and find out some interesting information about it-why do people research this gene, ect. Finally, a paragraph on the purpose of this lab, what were you tasked with discovering/what was the question you were asking? What was your initial hypothesis (this may change depending on your original hypothesis-that is okay)

Methods: Clearly describe the procedure (How did you set up each (both F1 and F2) of your crosses? How did you make maintenance plates? What strains did you use?, What types of plates did you use and what are the differences between them? etc.) You do not need to state the minutia, just the information that would be required for someone to be able to repeat your experiment. If you didn’t set up your F2 then state how it was set-up-ask if you are unclear.

Results and Figures: Present your findings in writing and with figures. All figure labels must be clear and neat. Each figure must have a title and a legend. You must include 1) Punnett Squares of your F1 and F2 crosses and 2) tables with your F1 and F2 data. DO NOT just put all the data in one spot and then talk about it somewhere else. The prose of the results should introduce the experiment a bit and describe why you set-up the cross the way you did and then you should show the results for that experiment. Describe the results while referencing the data table or figure. Then move on to the next experiment and repeat. DO NOT discuss the importance of the data here that is for the next section. The results section is very boring.

Discussion: Discuss the significance of your results. Did your data support your hypothesis? Why or why not? Discuss any sources of error.

References: Properly cite all of your work. Use only appropriate sources. Format in text citations and citation list correctly. I don’t care what format you use, just be consistent-APA or ACS are fine with me. You should have at least three peer-reviewed references.

 

Rubric:

Topic		Excellent	Good	Poor
Introduction	Background information	Background information clearly explains rationale for questions and experimental design. References are specific and appropriate to discussion points. Explains how findings from current experiment might contribute to understanding the topic in a larger context.	Some relevant context, but one dimensional.  Background with minor omissions. References too few.	Background info or references are narrow, inaccurate, or irrelevant. Rationale is vague.
	Question/Aim of the study	Clear and novel, based on literature review and personal observations	Based on understanding of given material.	Not clear or  specific
	Hypothesis	Simple direct statement that is testable and novelDescribes relationship between variables to be manipulated and outcomes to be measured. Brief predictions of different possible outcomes and significance.	Derived from understanding material, but may address multiple explanations or be vague.	Not simple and direct.  Not testable or worthy of testing.
Materials & Methods	Description	Concise and thorough. Written in third person, past tense. Includes detailed account of reagents, apparatus, software, measurements, timelines and protocols. Allows experiments to be reproducible.	Instructive, but too wordy. Minimal use of first person.	Vague. Not reproducible due insufficient details e.g. measurements
	Experimental design	Tests hypothesis, is replicated for multiple determinations of results, and contains controls with proper explanation of their importance.	Controls and replication are appropriate, but not thoroughly explained.	No replication. Controls are vague or irrelevant. Does not address question
Results	Prose	Data are written in the order in which experiments in the Methods were described. Data for all experiments are included and allow the reader to evaluate the conclusions.	Data are complete and address hypothesis.	Some data missing or confusing.
	Analysis / Statistics	Analysis is appropriate and concisely and objectively described with little interpretation. Analysis should state relationships between variables and measured outcomes only, including trends and patterns.	Analysis appropriate, but not well described or with inappropriate interpretation.	Analysis missing or inappropriate.
	Figures and Tables	Graphs are well-labeled with axes, units and legends. Graphs, figures and tables have titles, captions and are numbered for reference.	Appropriate format.  Contain only minor mistakes that do not interfere with meaning.	Contains errors or missing pieces (unlabeled axes, no title etc.).  Hard to derive meaning.
Discussion	Results Interpretation	Conclusion about whether to support or reject hypothesis are justified by the data.  Connections between hypothesis, data, and conclusions are comprehensive and  relevance of trends/outliers is also discussed	Conclusions are drawn from the data. Connections are logical.	Conclusions are not bound by data. Connections between hypothesis, data, and conclusions are weak
	Relevance/Next steps	Generates new insight. Suggests novel findings that would fill gaps in current knowledge and explains significance of findings to the field. Proposes further questions that need to be addressed in future experiments.	Useful, but indicates incomplete knowledge of the field or implications.	Vague and trivial or too broad and irrelevant. Not connected to the data.
	Error Analysis	Takes possible sources of error into account and also offers scientific explanations for findings different from expected. Discusses future procedures to reduce errors.	Not comprehensive, but more thoughtful than “human error”.	Trivial (e.g., “human error”). Inability to determine alternative reasons for data.
Citations	In text	Correctly inserted into text to fully frame the background, Methods, Discussion in current knowledge.  Justify text and make connections.	Proper format. Provide background. May be too few.	Improper format or irrelevant/misplaced.
	Bibliography	Proper format (APA style). Includes mostly primary literature and reviews.	Proper format, minor mistakes. Some primary literature.	Improper format. References to textbooks or websites.
Writing Quality	Organization / style	Organization according to the sections described in this rubric is maintained Professional style.	Organization is logical. Scientific style. Some use of colloquial expression.	No or some organization (does not flow).  Inappropriate style.
	Grammar	Correct grammar and spelling. Written in 3rd person, past tense	Few mistakes in spelling and grammar.	Errors hinder/prevent understanding. Poor spelling, incomplete sentences.
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Lab procedure

Lab 1- Working with C. elegans Objective- 1. Understand how to use a stereomicroscope. 2. Be able to move worms from one plate to another without gouging plates or killing worms. IntroductionThe first three weeks of lab are to get you acquainted with working with a dissecting scope, developing good hand-eye coordination while working with a dissecting scope and help you feel confident working with C. elegans (our genetic model organism). Today we will be reviewing how to use a stereomicroscope. You will be using this microscope for the vast majority of the semester so it is critical that you know how to properly use the microscope and do proper maintenance on it. Be sure to read over the Stereomicroscope Guide prior to lab. The second objective today is to work on moving worms from one plate to another plate. You will be doing this repeatedly in lab to set up mating plates. The major challenges when moving worms to different plates is two-fold: (1) gouging holes into the plate and (2) killing your worms. Worms like to tunnel, so if you gouge holes into your plates, the worms tunnel into the plates and you will not be able to get them again. The worms are pretty hearty but pressing down on them too hard will kill them…dead worms cannot mate. Today, your major goal is to get a feel for moving one worm at a time using an instrument called a worm pick. There are two types of worm picks available in this lab, those that look like a hockey stick and those with a loop on the end. Materials needed 1. Stereomicroscope Guide 2. Stereo/Dissecting Microscope and light source 3. One plate of worms 4. Three seeded 1x NGM plates 5. Worm pick 6. Gas flame Make sure to have all of your materials prior to beginning. Directions- 1. Follow the directions found in the Stereomicroscope Guide to first properly set-up and focus on your plate of worms. Once you feel comfortable locating your worms and zooming in and out move on to the next step. 2. Obtain a worm pick. Worm picks are made of platinum wire for two main reasons: it is easily pliable (can be shaped relatively easily-so if your pick becomes misshapen we can easily fix it) and it can heat up and cool down very fast (unlike the loops used in microbiology). 3. First you need to get a feel for how soft you need to be with your worm pick because you want to reduce plate gouging. Obtain a 1x NGM plate and place it on the stage. 4. Locate the edge of the E. coli and have the zoom at 20x. 5. Bring your worm pick into view. Move the worm pick up and down and touch the plate. Play around and determine how much pressure causes gouging and how much does not. When working with worms later, you do not want to gouge so play around now to figure it out. 6. Now zoom in to 45x and find your pick. Move it up and down and again touch it to the plate. When picking and moving worms you will likely want to zoom into 45x to see the worms well and then zoom out to place worms; so you should play around with adjusting the zoom and finding your pick at different magnifications. a. Once you feel comfortable with your hand-eye coordination under the scope and the amount of pressure required to NOT gouge the plate, throw away the plate you have been playing with (it is likely contaminated at this point) and move on to the next section. 7. Two methods for picking worms in this course. a. Scoop and Plop b. Single Worm Picking You will practice both of these techniques today. 8. For either method you will first put some E. coli onto the bottom of your pick from a seeded NGM plate (this will be your DESTRUCTION plate) and then use this E. coli to pick up a worm. The E. coli makes a sticky substance on the bottom of the pick, which allows you to pick up the worm and transfer it to another plate. a. Flame the pick briefly…you should not hold the pick in the flame for more than 1 or 2 seconds as it heats up and kills everything very quickly. Over flaming will reduce the life of the platinum wire and thus the pick. b. Open the lid to one of your 1x NGM seeded plates (not the worm plate). This plate will be your “destruction plate.” Use only this plate to put E. coli onto your pick. The other 1x NGM plate will be used to transfer worms to-It is important to keep your “destruction plate” separate from your “transfer plate.” Since you will not be putting worms onto your destruction plate, it is okay to gouge it and in fact, you will gouge it on purpose. Once your pick has been heated, we want to make sure it is cool before touching E. coli or the worms (if it is too hot it will kill both). To cool the pick touch it to a region of the plate without any E. coli. This will cool the pick and likely gouge the plate. Now swipe the pick through the E. coli (you will only need a little amount). The Scoop and Plop: c. Take one of your other seeded 1X NGM plates and label it “him-8” put your initials and the date as well. This is the plate you are going to use to transfer a large quantity of worms to. d. Looking through your dissecting scope at ~30X, isolate an area that has a lot of worms. Using your pick, gently swipe your pick through the area trying to get as many worms in the E.coli on your pick as possible. Now close the lid on that plate and get the plate you just labeled and put it under the scope. Move the plate under the scope until you see the edge of the E. coli now put your pick into the viewing area and gently place the worms onto the plate, without gouging the plate. e. Repeat this as many times as you need to get ~20 worms onto the plate. Picking a Single Worm: f. Repeat a-b. g. Now looking through your dissecting scope, isolate a worm that you want to pick. Using your pick, very gently move your pick over the worm so that you can see the pick in the scope. Gently touch the pick on top of the worm. You should be able to see a glob of E. coli on the bottom of the pick. Place the glob on the pick on top of your worm and use a small check motion to pick up the worm. Keep trying until you get the worm onto the pick. Try hard NOT to gouge your worm plate. h. Close the lid on your worm plate and remove it from the stage. i. Zoom out slightly and pick up your transfer plate, put it on the stage and open the lid. Find the edge of the E. coli and focus on it; this will help you be able to see where you are going to place your worm. Now, bring the pick with your worm under the scope and slowly lower it to the plate in a region near the E. coli but not directly in it. Using a check motion, place the worm onto the transfer plate. j. Repeat this until you have successfully transferred 5 individual, live worms. It is important that you work on transferring only one worm at a time! When setting up mating plates you will only want very specific worms and if you transfer too many or the wrong type it will confuse your data.

Worms Week 2- Sex determination and mutant worms Objectives 1) to tell the difference between males and hermaphrodites. 2) to be able to distinguish between an adult hermaphrodite and a larval stage hermaphrodite. 3) to distinguish between the four different mutant strains. Because C. elegans are hermaphroditic they can reproduce without a traditional mating event, therefore if you want to set up a successful genetic cross you must choose a hermaphrodite that has not yet become sexually mature, a larval stage four (L4). This will help ensure that the cross you set-up will result in the expected outcome. Below are pictures of a male (A), adult hermaphrodite (B) and L4 hermaphrodite (next page). To distinguish between a male and a hermaphrodite you must first be able to determine the head from the tail. To find the head on a worm, look for the pulsating pharynx. The pharynx is used to eat food and is located near the head of the worm, thus the opposite end of the worm is its tail. The structures of the tail are very different between males and hermaphrodites. At first this difference may seem very subtle, but overtime you will find the differences very obvious and easily be able to differentiate between males and hermaphrodites. Hermaphrodites have a “wispy” thread-like structure on their tail. The males have a blunt-ended tail that has a fan on it. See the pictures below for comparison. Distinguishing between adult hermaphrodites and L4 hermaphrodites is more challenging, but the easiest characteristic to check first is if there are any embryos in the worm. If you see large circular structures in the mid-section of the worm then she has embryos and is considered a sexually mature adult hermaphrodite. You DO NOT want this worm on your mating plate. The most obvious difference between adult hermaphrodites and L4s is at the vulva. The vulva in L4s is not completely matured and appears as a light spot near the middle of the worm. See the picture below. Using the contrast on the scope will help you locate this spot; it will take a bit of looking to see the difference between an L4 and an adult, but it is important that you can distinguish them. As worms mature through each larval stage and eventually become adults they grow larger, therefore a slightly smaller hermaphrodite is more likely to still be in the larval stage. It is okay to choose a worm that is an L3, but anything younger will not work in your matings. If you are having considerable trouble finding the light spot, you may pick a smaller worm, but beware, if you pick one too young your matings will not work well. Procedure 1- Making maintenance plates (these will be used next week to set-up any re-do crosses) 1. Obtain 2 maintenance plates: one him-8 strain and one mutant strain from the back bench. 2. Each student will need a total of two maintenance plates, one plate will be your stock him-8 strain and the other will be your stock mutant strain. 3. Label a new maintenance plate with your initials the date and the strain name. 4. Use the “scoop and plop” method to move several (at least 20) worms from the him-8 stock plate you made last week to your new him-8 plate. 5. Use the “scoop and plop” method to move several (at least 20) worms from the mutant lab stock plate to your new mutant plate. 6. Over the next few days you should research your mutant strain and hypothesize a pattern of inheritance and write out how you plan to determine the pattern of inheritance for the mutant strain you chose. Procedure 2- Sexing C. elegans The him-8 strain is a strain of worms that has a mutation in the HIM-8 gene which causes an increased rate of non-disjunction of the X-chromosome allowing for approximately 40% males and 60% hermaphrodites. You will use this plate today to find and transfer five males and five L4 hermaphrodites. 1. Label one maintenance plate males and the other hermaphrodites. Transfer 5 males to the male plate and 5 L4 hermaphrodites to the hermaphrodite plate. This week you must practice transferring one worm at a time as this will be critically important next week. Have your instructor look at your plates at least once to double check you. 2. Once you are done, tape your plates together and label them with your name and section. 3. Leave the lab stock plate of the him-8 strain on your bench, your instructor will collect them. 4. Once you are done, tape your maintenance plates together with the original plates and label them with your name. Tape your male and hermaphrodite plates together and label them with your name as well.

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