Department of Biological and Environmental Sciences BIO L320 – GENETICS LAB Dr. Jacqueline Jones Laboratory 1 – Mendelian Genetics – Introduction to Drosophila and Genetic basis of the Principle of Segregation in F2 progeny of a monohybrid cross. In this lab we will become familiar with the fruit fly Drosophila melanogaster that is used for conducting various genetic crosses in the laboratory. We will examine some of the morphological features of Drosophila that show variation and set up a monohybrid cross to show Mendel’s principal of segregation. We will also set up a cross involving a sex-linked trait. Because the Drosophila experiment will take 10 days per generation. Introduction to Drosophila We will practice handling and examining Drosophila fruit flies. We will follow the procedures from Mertens and Hammersmith – Investigation 1, with some important modifications. We will use the Carolina Formula 4-24 Instant Drosophila Medium. We will anesthetize flies by cooling 2 (to 2.5) minutes in freezer (no longer or else they die or become sterile) in a vial without food and keep them cool on ice packs rather than using a chemical anesthetic such as ether. Flies are cultured at 20-25°C in the incubators provided (these are preset at the required temperature and require no adjustment). Techniques for handling Drosophila Anesthetization of flies: Flies must be anesthetized in order to keep them inactive during examination or while they are being transferred into culture vials for mating. You have been provided with plastic dishes with ice, plastic containers for ice water baths, plastic petri dishes and white filter paper. Use the following procedure to anesthetize the flies: 1. Obtain a plastic dish with ice and place a petri dish with one piece of filter paper in it on the ice surface. 2. Shake the flies down into the bottom of the culture vial by tapping the vial, then remove the plug and quickly transfer the flies into an empty vial. 3. Insert the vial with the transferred flies into the freezer. 4. When the flies in the vial have stopped moving about 2 minutes (check them every 15 sec after 2 minutes), and then pour a few of them out onto the filter paper in the petri dish. 1 Do not leave flies in a freezer for more than a 2.5 minutes because they may die or become sterile!!! 5. Flies will normally remain anesthetized if kept on an ice bath or block. Culture vial preparation: 1. Obtain clean culture vial and foam plug. 2. Add 1 small scoop of dry food to vial. 3. Add 1 small scoop of tap water to vial. 4. Add two or three (2 or 3) grains of dry yeast to the vial. 5. Dry off any water on inside exposed walls of vial with a Kim-wipe. 6. Place foam plug in the vial to keep wandering Drosophila out. Setting up cultures: 1. Anesthetize flies. 2. Transfer flies to an empty vial with brush. 3. After the flies wake up they can then be added to the food vials. 4. Label vial with date and types of male and female flies used. 5. Subculture the mutant and wild stocks every one to two weeks; never keep the vials with cultures longer than 3 weeks. Setting up reciprocal crosses: 1. Clear adults from vials once pupal cases form. (Subculture if needed) 2. Collect virgin females within six to seven hours of clearing vials. 3. Place virgin females in vial with food. (Up to 8 per vial, based on availability) 4. Add males of other stock; try to wait before adding males so the females will be strong; use slightly less males than females. 5. Label vials properly. Two vials for each reciprocal cross are recommended. 6. Clear adults when pupal cases appear. (Set up in another vial if desired.) 7. Wait for F1 to emerge and score. 8. Collect data on the first 100 Fl's flies to emerge from each reciprocal cross. 9. Tabulate information on the Fl generation flies with regard to sex and phenotype for each reciprocal cross. 10. If conducting testcrosses, then F1 virgin females need to be collected. 11. When setting up the F1 flies for the F2 generation set up a new labelled vial then add an equal number of F1 males and females. The female flies do not need to be virgins, because it does not matter if mating occurred before or after the flies are put into the new vials as this is a sibmating. 12. Once again, clear the adults when the pupal cases appear. 13. Collect data on the first 100 F2 generation flies to emerge from each reciprocal cross. (14) Tabulate information on the F2 generation flies with regard to sex and phenotype for each reciprocal cross. Keep records of your crosses in Table 1.2 on (page 9-new book) (page 8 old book), or you can set up the information for in a MS Word file. Keep your records on the methods and data for your crosses for the F1 and F2 generation to write up later on. 2 Examination of sex and fly morphological characteristics Examine the preserved flies and note differences between males and females (See Figs. 1.2 and 1.3 in lab book and 11 and 12 in Carolina Drosophila Manual). You will be given some wild-type flies and five unknown mutant strains. Use the figures in the Carolina Drosophila manual to help you identify the phenotypes of the flies. Document the characteristics of these flies for the information requested in Table 1.1 on (page 9-New book) (page 6-old book), you will need to add a column at the end for the fifth mutant. Answer the three questions at the bottom of page(s) (9-10 new book/page 7 old book). You will then be asked to conduct reciprocal crosses between a wild type and a mutant strain of flies. • For the write-up of this lab, present the information requested in Table 1.1 and the three questions at the bottom of page 7 (old) page 8/10 (new book). You can either use the pages in the lab book or type up your answers and results. You must also include your own drawing/discussion on how to tell a male from female and how to prepare food, set up crosses, and anesthetize the flies. The write must be in your own words. Plagiarism will result in an F. This lab is worth a total of 50 points. 3 TROY UNIVERSITY BIO L320 Genetics Lab HOW TO WRITE A SCIENTIFIC LAB REPORT GENERAL IN APA FORMAT: Scientific laboratory reports or any other APA style paper written at a collegiate level have a standard format. The following are standard for APA papers:  1 inch margins on all sides  12-point font and the font style should be either Arial, or Times Roman  Some type of Header (Running Head:)  Numbered pages except the Title Page  Paragraphs indented 1-tab space  DOUBLE-SPACED throughout the paper including Title Page and Reference Page. Scientific laboratory reports have a separate Title Page and a separate Reference page. scientific reports have HEADINGS on each section of the report. These headings are:  Introduction  Materials & Methods  Results  Discussion TITLE: The first page of the lab report is the TITLE page. Do NOT number the Title page unless instructed to do so. The title of the lab report should NOT be too general but should reflect more specifically about the experiment. For example: “Fingerprinting” is too general for the title but instead, use something like: “Fingerprinting as an Effective Forensic Tool in Solving Murders.” The title should be informative; it should not be “cute.” A Running head is needed as a Header on all pages including the title page. The Running Head should appear as Running Head: SHORT TITLE OF PAPER IN ALL CAPS (but not in bold). Compiled by Rashmee Silwal for use in BIO L320 TROY UNIVERSITY NOTE: The title is in Title case (first letter of each word is capitalized except nonessential words.)! The Title page information should be absolute centered on the page. The Title page should NOT be numbered! (unless instructed). The Title page must be doubled-spaced, have a center indentation and include the following information: Title Lab Number Your Name (author) Names of those in the experimental group (do not put “Group Members” next to names) Instructor Class name Name of Institution Date of submission Example: Laboratory II – Mendelian Genetics – Genetic basis of the Principle of Segregation in F2 progeny of a monohybrid cross LAB 1 Group Members: Jane College, Harry Potter, Norman Rockwell and Barry Allen Dr. Jacqueline Jones/ Ms. Rashmee Silwal Genetics Lab Biol320 (A/B) August 15, 2017 Compiled by Rashmee Silwal for use in BIO L320 TROY UNIVERSITY Introduction The paper must be written in THIRD person. Do NOT use I, me, we, them, they, he, she or us. The Introduction does just that: INTRODUCES the topic or concept on which an experiment was performed. It is in the Introduction where you explain the ideas or concepts and NOT the experiment. Explain why the topic is important and if needed, a short background. How does the topic relate to your experiment? You don’t have to make it too wordy if you can introduce your topic correctly. You will need to cite at least two references in your Introduction section. ALL resources (references) MUST be properly CITED in the Introduction. Correct in text citations for the APA format must be present. Helpful links are posted on your course syllabus in Required Textbooks and Supplementary Materials. It is considered PLAGIARISM if credit is not given. Always Paraphrase. Avoid using direct quotes. In the Introduction, NEVER:  Discuss results  Write in first person  Include information just to fill in space.  State how data were collected (data are plural!)  Use numerous quotes instead of paraphrasing. But, avoid paraphrasing that isn’t written in your own words!  Plagiarize literature reviews or anything else in the paper (use quotations if directly quoting otherwise paraphrase or use your own words)  Start the Introduction with “In this experiment…” or “We experimented on….”  Include unnecessary information. “A Sharpie was used…”  Be wordy!  Triple space between paragraphs or next Heading  Start the Hypothesis with “I hypothesize… or “My group hypothesizes…” or anything else that is first person.” Compiled by Rashmee Silwal for use in BIO L320 TROY UNIVERSITY The last paragraph in the Introduction is for stating the Purpose and Hypothesis. As it is a part of introduction, you don’t need a title for this. It’s just the end sentence or section on Introduction. The Purpose is stating why the experiment was performed. Do NOT make statements such as: “The purpose of this experiment is to learn more about solutions.” Or, “The experiment will help me learn more about fingerprinting.” Or better yet, “It’s part of my grade.” The purpose should be logical and scientific. Basically, what was the lesson learned? Materials and Methodology This section is used to describe HOW the experiment was performed. There should be enough information so that another researcher or layperson can repeat the experiment and get the same or similar results. This section is written in sentence (narrative) format DESCRIBING the materials and/or equipment used and EXPLAINING the steps taken to collect data. You should NEVER just LIST materials used and steps taken. These should always be explained in a narrative, sentence format. DO NOT LIST!!! Do not plagiarize information from the lab manual or experiment handout! This section should always be written in PAST TENSE since you have already completed the experiment. Remember that you are describing what you did or how you set up the equipment so that data was collected and what materials were used. However, you must include enough detail that another person could set up the experiment the same way you did! Do NOT make any statements about the data collected. This will be in the Results section. Do not include unnecessary information such as: “A blue sharpie pen was used to label….” Or “Three marks were made on test tube #1…” Use statements such as: “Three mls of water and 3mls of methylene blue were added to each of three test tubes marked 1-3.” Results This section is for PRESENTING the data that were collected in the experiment. Your presentation of the data should allow readers to draw some type of conclusion about your experiment. You MUST include Tables and Figures (graphs) in this section about the data you collected. All tables and figures should be described in NARRATIVE format. You MUST describe the data in a narrative form and not just insert a table or graph or some type of listing. Follow the sentence descriptive form as with any section of the lab report except you will also insert a table(s) and Compiled by Rashmee Silwal for use in BIO L320 TROY UNIVERSITY figure(s) displaying the mathematical data. You should NEVER DISCUSS what the data MEAN in this section. Other readers should be able to easily read and understand what was measured from the table and figure. TABLES AND FIGURES MUST BE NUMBERED and have an informative title. The number and title are placed ABOVE the Table or Figure and double-spaced. The information inside the table is not double-spaced. Figures are also numbered and can include Graphs, Charts and Illustrations Figures (Graphs, Charts and Illustrations) must have Legends, Specific Titles, X & Y axes named, numbered and a short explanation of the figure. Discussion In this section, you will EXPLAIN or analyze the results. The hypothesis(es) should be restated. Your conclusions should be well organized and thoughts not scattered about in different paragraphs. You can also in-text cite references again in the discussion to support your thoughts. In this section, your results are INTERPRETED! Why are the results the way they are? Are the data similar to previous experiments? INTERPRET! Were your group results similar to other groups? Why or why not? Include a short discussion as to whether the data SUPPORTED or did NOT SUPPORT your hypothesis. A hypothesis cannot be Right or Wrong! It cannot be correct or not correct or good or bad. A hypothesis can ONLY be supported or not supported by the data collected. Do not make statements in your discussion such as “The results showed that my hypothesis was right (or wrong).” There is no right or wrong! If your hypothesis is not supported by the data, it does not necessarily mean that you did something wrong. You may have not done the experiment correctly but more than likely, there were other factors that may have altered the outcome. Discuss whatever you may think was a factor and it went wrong. Assumptions about any possible ERRORS while collecting data should be discussed. Refer to the data to support your speculations about the experiment. Do not state that “something went wrong” or “I was not clear about the instructions.” Compare your group data to those of other groups to see if your data are similar to the other groups. Compiled by Rashmee Silwal for use in BIO L320 TROY UNIVERSITY The last paragraph should state the major findings of the experiment/study. References References are the scientific/scholarly articles from VALID and RELIABLE sources about similar studies. NEVER use WIKIPEDIA as a reference. It is NOT valid or reliable!! References for Scientific papers should be listed in APA (American Psychological Association Writing Journal) format. The best way to gather your references is to do a database search in the Troy Library resources or Goggle Scholar first. A book is another great source that can be cited. Other reliable web sites include WebMD.com, CDC.gov, nih.gov, or nih.gov/PubMed. The Reference Heading should be centered at the top of the page. The heading should NOT be in bold, italicized or underlined. References are never bulleted or numbered. References should be listed on a separate page and in alphabetical order. Basic Format for List of references at the End of the Lab Report : APA style dictates that authors are listed last name first followed by first name initials; publication year goes between parentheses, followed by a period. The title of the article is in sentence-case, meaning only the first word and proper nouns in the title are capitalized. The periodical or journal title is italicized and in title case (first letter of each word is capitalized except non-essential words such as in, of, a, and, etc…), and is followed by the volume number which, with the title, is also italicized. If there is an issue number, it is in parentheses next to the volume number and is not italicized. Page numbers are in the format xx-xxx. If the article was retrieved from the internet, include the URL. It should be written as Retrieved from then the web address. Author, A. A., Author, B. B., & Author, C. C. (Year). Title of article. Title of Periodical, volume number (issue number not italicized), pages. Retrieved from http://www.xxxx.xxxx For more help, its best to refer the Supplementary Materials listed on the Course Syllabus. Compiled by Rashmee Silwal for use in BIO L320 C. Making Crosses when making a cross between two varieties, consider only those characters in which the parent flies differ. For example, in crossing flies having ebony body color with those having white eyes, only body color and eye color need to be observed carefully Secure anesthetized male flies of one variety and anesthetized virgin females of the other. While hold- ing the culture boule on its side, place these files in the bottle. Be sure to add some dry pevast granitules or perist spension to the medium before introducing the flies. Keep the bottle on its side until the flies have recovered from anesthetization. This position will prevent their becoming stuck in the medium. Label the bonle and recoed data in Table 1.2. After 7 or 8 days, remove the parent flies (P) to prevent their being confused with or mated with their offspring Flies of the first filial generation (FD) will soon begin to emerge After several F, flies have appeared, anesthetize and examine them, especially with regard to the characters by which the P, flies differed. Record in Table 1.2 the phenotypes of the F, flies of each sex: place these F1 flies in a fresh botle of medium. (Be sure to label the bottle! This mating will allow for the production of a second filial generation (F2). It is not necessary that the F, female flies be virgins for this mating 1. Why? The instructor may wish to have you testcross an F, female fly. In this case you should use a virgin female 10 Drosophila and Maize Experiments in Genetics: Monohybrid and Dihybrid Crosses 2. Why is a virgin female needed in this case? 3. What is a testcross? Trait 2 Unknown I 3 4 Unknow2 | Unknow? 5 5 Unknow 4 BOX No. 1 Wild Type Body color Brown Eye color red Brown yellow ebony ebony yellow wild white white wild Eye shape round round bar round bar Wing shape and size normal wild wild wild vestigial wild Antenna shape and size normal Brittle shape and size, normal shorter wild longer stubble stubble wild spineless Unknowb ebony wild round wild Shorter stubble